Microplate thermal shift assay apparatus for ligand development and multi-variable protein chemistry optimization

ABSTRACT

The present invention provides an assay apparatus for that includes a temperature adjusting means for simultaneously heating a plurality of samples, and a receiving means for receiving spectral emission from the samples while the samples are being heated. In further aspects of the invention, the receiving means can be configured to receive fluorescent emission, ultraviolet light, and visible light. The receiving means can be configured to receive spectral emission from the samples in a variety of ways, e.g., one sample at a time, simultaneously from more than one sample, or simultaneously from all of the samples. The temperature adjusting means can be configured with a temperature controller for changing temperature in accordance with a pre-determined profile.

[0001] This application claims priority to U.S. provisional applicationNo. 60/017,860, filed May 9, 1996, the entirety of which is incorporatedby reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSOREDRESEARCH AND DEVELOPMENT

[0002] Part of the work performed during development of this inventionutilized U.S. Government funds. The U.S. Government has certain rightsin this invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates generally to the screening ofcompound and combinatorial libraries. More particularly, the presentinvention relates to a method and apparatus for performing assays,particularly thermal shift assays.

[0005] 2. Related Art

[0006] In recent years, pharmaceutical researchers have turned tocombinatorial libraries as sources of new lead compounds for drugdiscovery. A combinatorial library is a collection of chemical compoundswhich have been generated, by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” asreagents. For example, a combinatorial polypeptide library is formed bycombining a set of amino acids in every possible way for a givencompound length (i.e., the number of amino acids in a polypeptidecompound). Millions of chemical compounds can theoretically besynthesized through such combinatorial mixing of chemical buildingblocks. Indeed, one investigator has observed that the systematic,combinatorial mixing synthesis of 100 million tetrameric compounds or 10billion pentameric compounds (Gordon, E. M. et al., J. Med. Chem.37:1233-1251 (1994)).

[0007] The rate of combinatorial library synthesis is accelerated byautomating compound synthesis and evaluation. For example,DirectedDiversity® is a computer based, iterative process for generatingchemical entities with defined physical, chemical and/or bioactiveproperties. The DirectedDiversitye system is disclosed in U.S. Pat. No.5,463,564, which is herein incorporated by reference in its entirety.

[0008] Once a library has been constructed, it must be screened toidentify compounds which possess some kind of biological orpharmacological activity. To screen a library of compounds, eachcompound in the library is equilibrated with a target molecule ofinterest, such as an enzyme. A variety of approaches have been used toscreen combinatorial libraries for lead compounds. For example, in anencoded library, each compound in a chemical combinatorial library canbe made so that an oligonucleotide “tag” is linked to it. A carefulrecord is kept of the nucleic acid tag sequence for each compound. Acompound which exerts an effect on the target enzyme is selected byamplifying its nucleic acid tag using the polymerase chain reaction(PCR). From the sequence of the tag, one can identify the compound(Brenner, S. et al., Proc. Natl. Acad. Sci. USA 89:5381-5383 (1992)).This approach, however, is very time consuming because it requiresmultiple rounds of oligonucleotide tag amplification and subsequentelectrophoresis of the amplification products.

[0009] A filamentous phage display peptide library can be screened forbinding to a biotinylated antibody, receptor or other binding protein.The bound phage is used to infect bacterial cells and the displayeddeterminant (i.e., the peptide ligand) is then identified (Scott, J. K.et al., Science 249:386-390 (1990)). This approach suffers from severaldrawbacks. It is time consuming. Peptides which are toxic to the phageor to the bacterium cannot be studied. Moreover, the researcher islimited to investigating peptide compounds.

[0010] In International Patent Application WO 94/05394 (1994), Hudson,D. et al., disclose a method and apparatus for synthesizing andscreening a combinatorial library of biopolymers on a solid-phase plate,in an array of 4×4 to 400×400. The library can be screened using afluorescently labeled, radiolabeled, or enzyme-linked target molecule orreceptor. The drawback to this approach is that the target molecule mustbe labeled before it can be used to screen the library.

[0011] A challenge presented by currently available combinatoriallibrary screening technologies is that they provide no information aboutthe relative binding affinities of different ligands for a receptorprotein. This is true whether the process for generating a combinatoriallibrary involves phage library display of peptides (Scott, J. K. et al.,Science 249:386-390 (1990)), random synthetic peptide arrays (Lam, K. S.et al., Nature 354:82-84 (1991)), encoded chemical libraries (Brenner,S. et al., Proc. Natl. Acad. Sci. USA 89:5381-5383 (1992)), the methodof Hudson (Intl. Appl. WO 94/05394), or most recently, combinatorialorganic synthesis (Gordon, E. et al., J. Med Chem. 37:1385-1399 (1994)).

[0012] To acquire quantitative binding data from the high throughputscreening of ligand affinities for a target enzyme, researchers haverelied on assays of enzyme activity. Enzymes lend themselves to highthroughput screening because the effect of ligand binding can bemonitored using kinetic assays. The experimental endpoint is usually aspectrophotometric change. Using a kinetic assay, most researchers use atwo-step approach to lead compound discovery. First, a large library ofcompounds is screened against the target enzyme to determine if any ofthe library compounds are active. These assays are usually performed ina single concentration (between 10⁻⁴-10⁻⁶ M) with one to threereplicates. Second, promising compounds obtained from the first screen(i.e., compounds which display activity greater than a predeterminedvalue) are usually re-tested to determine a 50% inhibitory concentration(IC₅₀), an inhibitor association constant (K_(i)), or a dissociationconstant (K_(d)). This two-step approach, however, is very laborintensive, time-consuming and prone to error. Each re-tested sample musteither be retrieved from the original assay plate or weighed out andsolubilized again. A concentration curve must then be created for eachsample and a separate set of assay plates must be created for eachassay.

[0013] There are other problems associated with the biochemical approachto high throughput screening of combinatorial libraries. Typically, agiven assay is not applicable to more than one receptor. That is, when anew receptor becomes available for testing, a new assay must bedeveloped. For many receptors, reliable assays are simply not available.Even if an assay does exist, it may not lend itself to automation.Further, if a K_(i) is the endpoint to be measured in a kinetic assay,one must first guess at the concentration of inhibitor to use, performthe assay, and then perform additional assays using at least sixdifferent concentrations of inhibitor. If one guesses too low, aninhibitor will not exert its inhibitory effect at the suboptimalconcentration tested.

[0014] In addition to the drawbacks to the kinetic screening approachdescribed above, it is difficult to use the kinetic approach to identifyand rank ligands that bind outside of the active site of the enzyme.Since ligands that bind outside of the active site do not preventbinding of spectrophotometric substrates, there is no spectrophotometricchange to be monitored. An even more serious drawback to the kineticscreening approach is that non-enzyme receptors cannot be assayed atall.

[0015] Thermal protein unfolding, or thermal “shift,” assays have beenused to determine whether a given ligand binds to a target receptorprotein. In a physical thermal shift assay, a change in a biophysicalparameter of a protein is monitored as a function of increasingtemperature. For example, in calorimetric studies, the physicalparameter measured is the change in heat capacity as a protein undergoestemperature induced unfolding transitions. Differential scanningcalorimetry has been used to measure the affinity of a panel ofazobenzene ligands for streptavidin (Weber, P. et al., J. Am. Chem. Soc.16:2717-2724 (1994)). Titration calorimetry has been used to determinethe binding constant of a ligand for a target protein (Brandts, J. etal., American Laboratory 22:3041 (1990)). The calorimetric approach,however, requires that the researcher have access to a calorimetricdevice. In addition, calorimetric technologies do not lend themselves tothe high throughput screening of combinatorial libraries, **threethermal scans per day are routine.

[0016] Like calorimetric technologies, spectral technologies have beenused to monitor temperature induced protein unfolding (Bouvier, M. etal., Science 265:398-402 (1994); Chavan, A. J. et al., Biochemistry33:7193-7202 (1994); Morton, A. et al., Biochemistry 1995:8564-8575(1995)). The calorimetric and spectral thermal shift studies describedabove all share a common limitation. In each study, only one bindingreaction was heated and assayed at a time. The single sample heating andassay configuration, as conventionally performed, has impeded theapplication of thermal shift technologies to high throughput screeningof combinatorial libraries. Thus, there is a need for a thermal shifttechnology which can be used to screen combinatorial libraries, can beused to identify and rank lead compounds, and is applicable to allreceptor proteins.

[0017] Thermal shift assays have been used to determine whether a ligandbinds to DNA. Calorimetric, absorbance, circular dichroism, andfluorescence technologies have been used (Pilch, D. S. et al., Proc.Natl. Acad. Sci. U.S.A. 91:9332-9336 (1994); Lee, M. et al., J. Med.Chem. 36:863-870 (1993); Butour, J.-L. et al., Eur. J. Biochem.202:975-980 (1991); Barcelo, F. et al., Chem. Biol. Interactions74:315-324 (1990)). As used conventionally, however, these technologieshave impeded the high throughput screening of nucleic acid receptors forlead compounds which bind with high affinity. Thus, there is a need fora thermal shift technology which can be used to identify and rank theaffinities of lead compounds which bind to DNA sequences of interest.

[0018] When bacterial cells are used to overexpress exogenous proteins,the recombinant protein is often sequestered in bacterial cell inclusionbodies. For the recombinant protein to be useful, it must be purifiedfrom the inclusion bodies. During the purification process, therecombinant protein is denatured and must then be renatured. It isimpossible to predict the renaturation conditions that will facilitateand optimize proper refolding of a given recombinant protein. Usually, anumber of renaturing conditions must be tried before a satisfactory setof conditions is discovered. In a study by Tachibana et al., each offour disulfide bonds were singly removed, by site-directed mutagenesis,from hen lysozyme (Tachibana et al., Biochemistry 33:15008-15016(1994)). The mutant genes were expressed in bacterial cells and therecombinant proteins were isolated from inclusion bodies. Each of theisolated proteins were renatured under different temperatures andglycerol concentrations. The efficacy of protein refolding was assessedin a bacteriolytic assay in which bacteriolytic activity was measured asa function of renaturing temperature. The thermal stability of eachprotein was studied using a physical thermal shift assay. In this study,however, only one sample reaction was heated and assayed at a time. Thesingle sample heating and assay configuration prevents the applicationof thermal shift technologies to high throughput screening of amultiplicity of protein refolding conditions. Thus, there is a need fora thermal shift technology which can be used to rank the efficacies ofvarious protein refolding conditions.

[0019] Over the past four decades, X-ray crystallography and theresulting atomic models of proteins and nucleic acids have contributedgreatly to an understanding of structural, molecular, and chemicalaspects of biological phenomena. However, crystallographic analysisremains difficult because there are not straightforward methodologiesfor obtaining X-ray quality protein crystals. Conventional methodscannot be used quickly to identify crystallization conditions that havehighest probability of promoting crystallization (Garavito, R. M. etal., J. Bioenergtics and Biomembranes 28:13-27 (1996)). Even the use offactorial design experiments and successive automated grid searches(Cox, M. J., & Weber, P. C., J. Appl. Cryst. 20:366-373 (1987); Cox, M.J., & Weber, P. C., J. Crystal Growth 90:318-324 (1988)) do notfacilitate rapid, high throughput screening of biochemical conditionsthat promote the crystallization of X-ray quality protein crystals.Moreover, different proteins are expected to require differentconditions for protein crystallization, just as has been the experiencefor their folding (McPherson, A., In: Preparation and Analysis ofProtein Crystals, Wiley Interscience, New York, (1982)). Conventionalmethods of determining crystallization conditions are cumbersome, slow,and labor intensive. Thus, there is a need for a rapid, high throughputtechnology which can be used to rank the efficacies of proteincrystallization conditions.

[0020] Rapid, high throughput screening of combinatorial molecules orbiochemical conditions that stabilize target proteins in thermal shiftassays would be facilitated by the simultaneous heating of many samples.To date, however, thermal shift assays have not been performed that way.Instead, the conventional approach to performing thermal shift assayshas been to heat and assay only one sample at a time. That is,researchers conventionally 1) heat a sample to a desired temperature ina heating apparatus; 2) assay a physical change, such as absorption oflight or change in secondary, tertiary, or quaternary protein structure;3) heat the samples to the next highest desired temperature; 4) assayfor a physical change; and 5) continue this process repeatedly until thesample has been assayed at the highest desired temperature.

[0021] This conventional approach is disadvantageous for at least tworeasons. First, this approach is labor intensive. Second, this approachlimits the speed with which thermal shift screening assays can beperformed and thereby precludes rapid, high-throughput screening ofcombinatorial molecules binding to a target receptor and biochemicalconditions that stabilize target proteins. Thus, there is a need for anapparatus capable of performing rapid, high-throughput thermal shiftassays that will be suitable for all receptors, including reversiblyfolding proteins.

SUMMARY OF THE INVENTION

[0022] The present invention provides a multi-variable method forranking the efficacy of one or more of a multiplicity of differentmolecules or different biochemical conditions for stabilizing a targetmolecule which is capable of denaturing due to a thermal change. Themethod comprises contacting the target molecule with one or more of amultiplicity of different molecules or different biochemical conditionsin each of a multiplicity of containers, simultaneously heating themultiplicity of containers, measuring in each of the containers aphysical change associated with the thermal denaturation of the targetmolecule resulting from heating, generating a thermal denaturation curvefor the target molecule as a function of temperature for each of thecontainers, comparing each of the denaturation curves to (i) each of theother thermal denaturation curves and to (ii) the thermal denaturationcurve obtained for the target molecule under a reference set ofbiochemical conditions, and ranking the efficacies of multiplicity ofdifferent molecules or the different biochemical conditions according tothe change in each of the thermal denaturation curves.

[0023] The present invention provides a multi-variable method foroptimizing the shelf life of a target molecule which is capable ofdenaturing due to a thermal change. The method comprises contacting thetarget molecule with one or more of a multiplicity of differentmolecules or different biochemical conditions in each of a multiplicityof containers, simultaneously heating the multiplicity of containers,measuring in each of the containers a physical change associated withthe thermal denaturation of the target molecule resulting from heating,generating a thermal denaturation curve for the target molecule as afunction of temperature for each of the containers, comparing each ofthe denaturation curves to (i) each of the other thermal denaturationcurves and to (ii) the thermal denaturation curve obtained for thetarget under a reference set of biochemical conditions, and ranking theefficacies of multiplicity of different molecules or the differentbiochemical conditions according to the change in each of the thermaldenaturation curves.

[0024] The present invention also provides a multi-variable method forranking the affinity of a combination of two or more of a multiplicityof different molecules for a target molecule which is capable ofdenaturing due to a thermal change. The method comprises contacting thetarget molecule with a combination of two or more different molecules ofthe multiplicity of different molecules in each of a multiplicity ofcontainers, simultaneously heating the multiplicity of containers,measuring in each of the containers a physical change associated withthe thermal denaturation of the target molecule resulting from theheating, generating a thermal denaturation curve for the target moleculeas a function of temperature for each of the containers, comparing eachof the thermal denaturation curves with (i) each of the other thermaldenaturation curves obtained for the target molecule and to (ii) thethermal denaturation curve for the target molecule in the absence of anyof the two or more different molecules, and ranking the affinities ofthe combinations of the two or more multiplicity of different moleculesaccording to the change in each of the thermal denaturation curves.

[0025] The present invention also provides a multi-variable method forranking the efficacies of one or more of a multiplicity of differentbiochemical conditions to facilitate the refolding of a sample of adenatured protein. The method comprises placing one of the refoldedprotein samples in each of a multiplicity of containers, wherein each ofthe refolded protein samples has been previously refolded according toone or more of the multiplicity of conditions, simultaneously heatingthe multiplicity of containers, measuring in each of the containers aphysical change associated with the thermal denaturation of the proteinresulting from heating, generating a thermal denaturation curve for theprotein as a function of temperature for each of the containers,comparing each of the denaturation curves to (i) each of the otherthermal denaturation curves and to (ii) the thermal denaturation curveobtained for the native protein under a reference set of biochemicalconditions, and ranking the efficacies of the multiplicity of differentrefolding conditions according to the change in the magnitude of thephysical change of each of the thermal denaturation curves.

[0026] The present invention also provides a further multi-variablemethod for ranking the efficacies of one or more of a multiplicity ofdifferent biochemical conditions to facilitate the refolding of a sampleof a denatured protein, which comprises determining one or morecombinations of a multiplicity of different conditions which promoteprotein stabililty, folding the denatured protein under said one or morecombinations of biochemical conditions that were identified as promotingprotein stabilization, asseessing folded protein yield, ranking theefficacies of said multiplicity of different refolding conditionsaccording to folded protein yield, and repeating these steps until acombination of biochemical conditions that promote optimal proteinfolding are identified.

[0027] Using the microplate thermal shift assay, one can determine oneor more biochemical conditions have an additive effect on proteinstability. Once a set of biochemical conditions that facilitate anincrease in protein stability have been identified using the thermalshift assay, the same set of conditions can be used in protein foldingexperiments with recombinant protein. If the conditions that promoteprotein stability in the thermal shift assay correlate with conditionsthat promote folding of recombinant protein, conditions can be furtheroptimized by performing additional thermal shift assays until acombination of stabilizing conditions that result in further increaseprotein stability are identified. Recombinant protein is then foldedunder those conditions. This process is repeated until optimal foldingconditions are identified.

[0028] The present invention also provides a multi-variable method forranking the efficacy of one or more of a multiplicity of differentbiochemical conditions for facilitating the crystallization of a proteinwhich is capable of denaturing due to a thermal change. The methodcomprises contacting the protein with one or more of the multiplicity ofdifferent biochemical conditions in each of a multiplicity ofcontainers, simultaneously heating the multiplicity of containers,measuring in each of the containers a physical change associated withthe thermal denaturation of the protein resulting from the heating,generating a thermal denaturation curve for the protein as a function oftemperature for each of the containers, comparing each of thedenaturation curves to (i) each of the other thermal denaturation curvesand (ii) to the thermal denaturation curve obtained using a referenceset of biochemical conditions, and ranking the efficacies of themultiplicity of different biochemical conditions according to the changein each of the thermal denaturation curves.

[0029] The present invention also provides a method for ranking theaffinity of each of a multiplicity of different molecules for a targetmolecule which is capable of denaturing due to a thermal change. Themethod comprises contacting the target molecule with one molecule of amultiplicity of different molecules in each of a multiplicity ofcontainers, simultaneously heating the containers, measuring in each ofthe containers a physical change associated with the thermaldenaturation of the target molecule resulting from heating, generating athermal denaturation curve for the target molecule as a function oftemperature in each of the containers, comparing each of the thermaldenaturation curves with the thermal denaturation curve obtained for thetarget molecule in the absence of any of the molecules in themultiplicity of different molecules, and ranking the affinities of eachmolecule according to the change in each of the thermal denaturationcurves.

[0030] The present invention also provides a method for assaying a poolor collection of a multiplicity of different molecules for a moleculewhich binds to a target molecule which is capable of denaturing due to athermal change. The method comprises contacting the target molecule witha collection of at least two molecules of a multiplicity of differentmolecules in each of a multiplicity of containers, simultaneouslyheating the multiplicity of containers, measuring in each of thecontainers a physical change associated with the thermal denaturation ofthe target molecule resulting from heating, generating a set of thermaldenaturation curves for the target molecule as a function of temperaturefor each of the containers, comparing each of the thermal denaturationcurves with the thermal denaturation curve obtained for the targetmolecule in the absence of any of the molecules in the multiplicity ofdifferent molecules, ranking the affinities of the collections ofdifferent molecules according to the change in each of the thermaldenaturation curves, selecting the collection of different moleculeswhich contains a molecule with affinity for the target molecule,dividing the selected collection into smaller collections of moleculesin each of a multiplicity of containers, and repeating the above stepsuntil a single molecule responsible for the original thermal shift inthe multiplicity of molecules is identified.

[0031] This invention also provides an improved method for generatinglead compounds which comprises synthesizing a multiplicity of compoundsand testing the ability of each compound to bind to a receptor molecule.The improvement comprises contacting the receptor molecule with onecompound of a multiplicity of different compounds in each of amultiplicity of wells in a microplate, simultaneously heating the wells,measuring in each of the wells a physical change, resulting fromheating, associated with the thermal denaturation of the receptormolecule, generating a thermal denaturation curve for the receptormolecule as a function of temperature in each of the wells, comparingeach of the thermal denaturation curves with the thermal denaturationcurve obtained for the receptor molecule in the absence of any of thecompounds in the multiplicity of different compounds, and ranking theaffinities of each compound according to the change in each of thethermal denaturation curves.

[0032] The present invention also provides a product of manufacturewhich comprises a carrier having a multiplicity of containers therein,each of the containers containing a target molecule which is capable ofdenaturation due to heating, and at least one molecule selected from amultiplicity of different molecules present in a combinatorial library,wherein each of the different molecules are present in a different oneof the multiplicity of containers in the carrier.

[0033] Optimization of protein stability, ligand binding, proteinfolding, and protein crystallization are multi-variable events.Multi-variable optimization problems require large numbers of parallelexperiments to collect as much data as possible in order to determinewhich variables influence a favorable response. For example,multi-variable optimization problems require large numbers of parallelexperiments to collect as much data as possible in order to determinewhich variables influence protein stabililty. In this regard, bothprotein crystallization and quantitative structure activity relationshipanalyses have greatly benefited from mass screening protocols thatemploy matrix arrays of incremental changes in biochemical or chemicalcomposition. Thus, in much the same way that quantitative structureactivity relationships are constructed to relate variations of chemicalfunctional groups on ligands to their effect on binding affinity to agiven therapeutic receptor, the methods and apparatus of the presentinvention facilitate the construction of a quantitative model thatrelates different biochemical conditions to experimentally measuredprotein stability, ligand specificity, folded protein yield, andcrystallized protein yield.

[0034] The present invention offers a number of advantages over previoustechnologies that are employed to optimize multi-variable events such asprotein stabilization, ligand binding, protein folding, and proteincrystallization. Foremost among these advantages is that the presentinvention facilitates high throughput screening. Further, the presentinvention offers a number of advantages over previous technologies thatare employed to screen combinatorial libraries. Foremost among theseadvantages is that the present invention facilitates high throughputscreening of combinatorial libraries for lead compounds. Many currentlibrary screening technologies simply indicate whether a ligand binds toa receptor or not. In that case, no quantitative information isprovided. No information about the relative binding affinities of aseries of ligands is provided. In contrast, the present inventionfacilitates the ranking of a series of compounds for their relativeaffinities for a target receptor. With this information in hand, astructure-activity relationship can be developed for a set of compounds.The ease, reproducibility, and speed of using ligand-dependent changesin midpoint unfolding temperature (T_(m)) to rank relative bindingaffinities makes the present invention a powerful tool in the drugdiscovery process.

[0035] Typically, the conventional kinetic screening approach requiresat least six additional well assays at six different concentrations ofinhibitor to determine a K_(i). Using the present invention, throughputis enhanced ˜6 fold over the enzyme based assays because one completebinding experiment can be performed in each well of a multiwellmicroplate. The kinetic screening approached are even further limited bythe usual compromise between dilution and signal detection, whichusually occurs at a protein concentration of about 1 nM. In this regard,the calorimetric approaches, either differential scanning calorimetry orisothermal titrating calorimetry, are at an even worse disadvantagesince they are limited to solitary binding experiments, usually 1 perhour. In contrast, the present invention affords a wide dynamic range ofmeasurable binding affinities, from ˜10⁻⁴ to 10⁻¹⁵ M, in a single well.

[0036] The present invention does not require radioactively labeledcompounds. Nor does it require that receptors be labeled with afluorescent or chromophoric label.

[0037] A very important advantage of the present invention is that itcan be applied universally to any receptor that is a drug target Thus,it is not necessary to invent a new assay every time a new receptorbecomes available for testing. When the receptor under study is anenzyme, researchers can determine the rank order of affinity of a seriesof compounds more quickly and more easily than they can usingconventional kinetic methods. In addition, researchers can detect ligandbinding to an enzyme, regardless of whether binding occurs at the activesite, at an allosteric cofactor binding site, or at a receptor subunitinterface. The present invention is equally applicable to non-enzymereceptors, such as proteins and nucleic acids.

[0038] In a further aspect of the present invention, an assay apparatusis provided that includes a heating means for simultaneously heating aplurality of samples, and a receiving means for receiving spectralemission from the samples while the samples are being heated. In yet afurther aspect of the present invention, an assay apparatus is providedthat includes a temperature adjusting means for simultaneously adjustinga temperature of a plurality of samples in accordance with apre-determined temperature profile, and a receiving means for receivingspectral emission from the samples while the temperature of the samplesis adjusted in accordance with the temperature profile.

[0039] In yet a further aspect, the present invention also provides anassay apparatus that includes a movable platform on which are disposed aplurality of heat conducting blocks. The temperature of the heatconducting blocks, and their samples, are adjusted by a temperatureadjusting means. Each of the plurality of heat conducting blocks isadapted to receive a plurality of samples. A light source is providedfor emitting an excitatory wavelength of light for the samples. Whilethe temperature of the samples is being adjusted, a sensor detects thespectral emission from the samples in response to the excitatorywavelength of light. The movable platform is moved between heatconducting blocks to sequentially detect spectral emission from thesamples in each of the plurality of heat conducting blocks.

[0040] The assay apparatus of the present invention affords the artisanthe opportunity to rapidly screen molecules and biochemical conditionsthat affect protein stability. Samples are simultaneously heated over arange of temperatures. During heating, spectral emissions are received.The assay apparatus of the present invention also provides the artisanwith an opportunity for conveniently and efficiently carrying out themethods of the present invention. The assay apparatus of the presentinvention is particularly adapted for carrying out thermal shift assaysof molecules and biochemical conditions that stabilize target proteins.

[0041] Because the apparatus of the present invention comprises both aheating means and a spectral emission receiving means, the apparatus ofthe present invention obviates the need to heat samples in one apparatusand transfer the samples to another apparatus prior to taking spectralemission readings. As a result, the apparatus of the present inventionfacilitates changing temperature in accordance with a pre-determinedtemperature profile, rather than incremental temperature increases andintermediate cooling steps. Thus, more data points can be collected fora given sample and more accurate information can be obtained.

[0042] Further, because the assay apparatus of the present inventioncomprises both a heating means and a spectral emission receiving means,spectral measurements can be taken from the samples while they are beingheated. Thus, using the assay apparatus of the present invention, theartisan can study both irreversibly unfolding proteins and reversiblyfolding proteins.

[0043] Further features and advantages of the present invention aredescribed in detail below with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

[0044] The present invention is described with reference to theaccompanying drawings. In the drawings, like reference numbers indicateidentical or functionally similar elements. Additionally, the left-mostdigit(s) of a reference number identifies the drawing in which thereference number first appears.

[0045]FIG. 1 shows the results of a microplate thermal shift assay forligands which bind to the active site of human α-thrombin (withturbidity as the experimental signal).

[0046]FIG. 2 shows the results of a microplate thermal shift assay forligands which bind to acidic fibroblast growth factor (aFGF) (withturbidity as the experimental signal).

[0047]FIG. 3 shows the results of a microplate thermal shift assay forligand binding to the active site of human α-thrombin (with fluorescenceemission as the experimental signal). The lines drawn through the datapoints represent non-linear least squares curve fits of the data usingthe equation shown at the bottom of the figure. There are five fittingparameters for this equation of y(T) vs. T: (1) y_(f), thepre-transitional fluorescence for the native protein; (2) y_(u), thepost-transitional fluorescence for the unfolded protein; (3) T_(m), thetemperature at the midpoint for the unfolding transition; (4) ΔH_(u),the van't Hoff unfolding enthalpy change; and (5) ΔC_(pu), the change inheat capacity upon protein unfolding. The non-linear least squares curvefitting was accomplished using KaleidaGraph™ 3.0 software (SynergySoftware, Reading Pa.), which allows the five fitting parameters tofloat while utilizing Marquardt methods for the minimization of the sumof the squared residuals.

[0048]FIG. 4 shows the result of a microplate thermal shift assay ofligands which bind to the D(II) domain of human FGF receptor 1 (D(II)FGFR1) (with fluorescence emission as the experimental signal). Thelines drawn through the data points represent non-linear least squarescurve fits of the data using the equation shown at the bottom of thefigure, as described for FIG. 3.

[0049]FIG. 5 shows the results of a miniaturized microplate thermalshift assay for Factor D in the absence of any ligands.

[0050]FIG. 6 shows the results of a microplate thermal shift assay forFactor Xa in the absence of any ligands.

[0051]FIG. 7 shows the results of a miniaturized microplate thermalshift assay of a ligand that binds to the catalytic site of humanα-thrombin.

[0052]FIG. 8 shows the results of a miniaturized microplate thermalshift assay of aprosulate binding to the D(II) domain of human FGFreceptor 1.

[0053]FIG. 9 shows the results of a miniaturized microplate thermalshift assay for urokinase in the presence of glu-gly-argchloromethylketone.

[0054]FIG. 10 shows the results of a miniaturized microplate thermalshift assay of human α-thrombin in which the assay volume is 2 μl.Thermal denaturation curves for three experiments are shown.

[0055]FIG. 11 shows the results of a miniaturized microplate thermalshift assay of human α-thrombin in which the assay volume is 5 μl.Thermal denaturation curves for five experiments are shown.

[0056]FIG. 12 shows the results of a single temperature microplatethermal shift assay of human α-thrombin in the presence of fourdifferent compounds in four separate experiments.

[0057]FIG. 13 shows the results of a microplate thermal shift assay ofthe intrinsic tryptophan fluorescence of human α-thrombin. In thisassay, blank well fluorescence was not subtracted from samplefluorescence.

[0058]FIG. 14 shows the results of a microplate thermal shift assay ofthe intrinsic tryptophan fluorescence of human α-thrombin. In thisassay, blank well fluorescence was subtracted from sample fluorescence.

[0059]FIG. 15 shows the results of microplate thermal shift assays ofsingle ligand binding interactions to three different classes of bindingsites for human α-thrombin.

[0060]FIG. 16 shows the results of microplate thermal shift assays ofmulti-ligand binding interactions for human α-thrombin.

[0061] FIGS. 17A-D show the results of microplate thermal shift assaysof the effect of pH and various sodium chloride concentrations on thestability of human α-thrombin. In FIG. 17A, the fluorophore is 1,8-ANS.In FIG. 17B, the fluorophore is 2,6-ANS. In FIG. 17C, the fluorophore is2,6-TNS. In FIG. 17D, the fluorophore is bis-ANS.

[0062]FIG. 18 shows the results of microplate thermal shift assays ofthe effect of calcium chloride, ethylenediaminetetraacetic acid,dithiothreitol, and glycerol on the stability of human α-thrombin.

[0063]FIG. 19 shows the results of microplate thermal shift assays ofthe effect of pH and sodium chloride concentration of the stability ofhuman D(II) FGF receptor 1.

[0064]FIG. 20 shows the results of microplate thermal shift assays ofthe effect of various biochemical conditions on the stability of humanD(II) FGF receptor 1.

[0065]FIG. 21 shows the results of microplate thermal shift assays ofthe effect of various biochemical conditions on the stability of humanD(II) FGF receptor 1.

[0066]FIG. 22 shows the results of microplate thermal shift assays ofthe effect of various biochemical conditions on the stability of humanD(II) FGF receptor 1.

[0067]FIG. 23 shows the results of microplate thermal shift assays ofthe effect of various biochemical conditions on the stability of humanD(II) FGF receptor 1.

[0068]FIG. 24 shows the results of microplate thermal shift assays ofthe effect of various biochemical conditions on the stability of humanD(II) FGF receptor 1.

[0069]FIG. 25 shows the results of microplate thermal shift assays ofthe effect of various biochemical conditions on the stability of humanurokinase.

[0070]FIG. 26 is a schematic diagram of a thermodynamic model for thelinkage of the free energies of protein folding and ligand binding.

[0071]FIG. 27 is a schematic diagram of a method of screeningbiochemical conditions that optimize protein folding.

[0072]FIG. 28 shows the results of microplate thermal shift assays ofhuman α-thrombin stability using various fluorophores.

[0073]FIG. 29 shows a schematic diagram of one embodiment of an assayapparatus of the present invention.

[0074]FIG. 30 shows a schematic diagram of an alternate embodiment ofthe assay apparatus of the present invention.

[0075]FIG. 31 shows a schematic diagram of the assay apparatus accordingto another embodiment of the present invention.

[0076] FIGS. 32A-E illustrate one embodiment of a thermal electric stagefor the assay apparatus of the present invention. FIG. 32A shows a sideview of the thermal electric stage. FIG. 32B shows a top view of thethermal electric stage. FIGS. 32C-E show three configurations of insertsthat can be attached to the thermal electric stage. In one embodiment,inserts accommodate a microtitre plate. In such an embodiment, assaysamples are contained within the wells of the microtitre plate.

[0077]FIG. 33 is a schematic diagram illustrating a top view of anotherembodiment of the assay apparatus of the present invention.

[0078]FIG. 34 is a schematic diagram illustrating the top view of theembodiment of the assay apparatus shown in FIG. 33 with a housinginstalled.

[0079]FIG. 35 is a schematic diagram illustrating a side view of theembodiment of the assay apparatus shown in FIGS. 33 and 34.

[0080]FIGS. 36A and 36B illustrate a temperature profile and how thetemperature profile is implemented using the automated assay apparatusof the present invention.

[0081]FIG. 37 shows an exemplary computer system suitable for use withthe present invention.

[0082]FIG. 38 shows a flow diagram illustrating one embodiment forimplementation of the present invention.

[0083]FIG. 39 shows a flow diagram illustrating an alternate embodimentfor implementation of the present invention.

[0084]FIG. 40 shows a comparison of the results of microplate thermalshift assays of human α-thrombin denaturation performed using afluorescence scanner and a CCD camera.

[0085]FIGS. 41A and 41B show photographs of microplate thermal shiftassay of human α-thrombin denaturation performed using a CCD camera.FIG. 41A: V-bottom well microplate. FIG. 41B: dimple microplate.

[0086]FIG. 42 shows a comparison of the results of microplate thermalshift assays of human α-thrombin denaturation performed using afluorescence scanner and a CCD camera.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0087] In the following description, reference will be made to variousterms and methodologies known to those of skill in the biochemical andpharmacological arts. Publications and other materials setting forthsuch known terms and methodologies are incorporated herein by referencein their entireties as though set forth in full.

Overview of the Methods of the Present Invention

[0088] The present invention provides a method for ranking amultiplicity of different molecules in the order of their ability tobind to a target molecule which is capable of unfolding due to a thermalchange. In one embodiment of this method, the target molecule iscontacted with one molecule of a multiplicity of different molecules ineach of a multiplicity of containers. The containers are thensimultaneously heated, in intervals, over a range of temperatures. Aftereach heating interval, a physical change associated with the thermaldenaturation of the target molecule is measured. In an alternateembodiment of this method, the containers are heated in a continuousfashion. A thermal denaturation curve is plotted as a function oftemperature for the target molecule in each of the containers.Preferably, the temperature midpoint, T_(m), of each thermaldenaturation curve is identified and is then compared to the T_(m) ofthe thermal denaturation curve obtained for the target molecule in theabsence of any of the molecules in the containers. Alternatively, anentire thermal denaturation curve can be compared to other entirethermal denaturation curves using computer analytical tools.

[0089] The term “combinatorial library” refers to a plurality ofmolecules or compounds which are formed by combining, in every possibleway fore given compound length, a set of chemical or biochemicalbuilding blocks which may or may not be related in structure.Alternatively, the term can refer to a plurality of chemical orbiochemical compounds which are formed by selectively combining aparticular set of chemical building blocks. Combinatorial libraries canbe constructed according to methods familiar to those skilled in theart. For example, see Rapoport et al., Immunology Today 16:43-49 (1995);Sepetov, N. F. et al., Proc. Natl. Acad. Sci. U.S.A. 92:5426-5430(1995); Gallop, M. A. et al., J. Med. Chem. 9:1233-1251 (1994); Gordon,E. M. et al., J. Med. Chem. 37:1385-1401 (1994); Stankova, M. et al.,Peptide Res. 7:292-298 (1994); Erb, E. et al., Proc. Natl. Acad. Sci.U.S.A. 91:11422-11426 (1994); DeWitt, S. H. et al., Proc. Natl. Acad.Sci. U.S.A. 90:6909-6913 (1993); Barbas, C. F. et al., Proc. Natl. Acad.Sci. U.S.A. 89:4457-4461 (1992); Brenner, S. et al. Proc. Natl. Acad.Sci. U.S.A. 89:5381-5383 (1992); Lam, K. S. et al., Nature 354:82-84(1991); Devlin, J. J. et al., Science 245:404406 (1990); Cwirla, S. E.et al., Proc. Natl. Acad. Sci. U.S.A. 87:6378-6382 (1990); Scott, J. K.et al., Science 249:386-390 (1990). Preferably, the term “combinatoriallibrary” refers to a DirectedDiversity library, as set forth in U.S.Pat. No. 5,463,564. Regardless of the mariner in which a combinatoriallibrary is constructed, each molecule or compound in the library iscatalogued for future reference.

[0090] The term “compound library” refers to a plurality of molecules orcompounds which were not formed using the combinatorial approach ofcombining chemical or biochemical building blocks. Instead, a compoundlibrary is a plurality of molecules or compounds which are accumulatedand are stored for use in future ligand-receptor binding assays. Eachmolecule or compound in the compound library is catalogued for futurereference.

[0091] The terms “multiplicity of molecules,” “multiplicity ofcompounds,” or “multiplicity of containers” refer to at least twomolecules, compounds, or containers.

[0092] The term “multi-variable” refers to more than one experimentalvariable.

[0093] The term “screening” refers to the testing of a multiplicity ofmolecules or compounds for their ability to bind to a target moleculewhich is capable of denaturing.

[0094] The term “ranking” refers to the ordering of the affinities of amultiplicity of molecules or compounds for a target molecule, accordingto the ability of the molecule or compound to shift the thermaldenaturation curve of the target molecule, relative to the thermaldenaturation curve of the target molecule in the absence of any moleculeor compound.

[0095] The term “ranking” also refers to the ordering of the efficaciesof a multiplicity of biochemical conditions in optimizing proteinstabilization, protein folding, protein crystallization, or proteinshelf life. In the context of optimization of protein stabilization,optimization of protein folding, optimization of proteincrystallization, and optimization of protein shelf life, the term“ranking” refers to the ordering of the efficacies of one or morecombinations of biochemical conditions to shift the thermal denaturationcurve of the target molecule, relative to the thermal denaturation curveof the target molecule under a reference set of conditions.

[0096] The term “reference set of conditions” refers to a set ofbiochemical conditions under which a thermal denaturation curve for atarget molecule is obtained. Thermal denaturation curves obtained underconditions different than the reference conditions are compared to eachother and to the thermal denaturation curve obtained for the targetmolecule under reference conditions.

[0097] As discussed above, ranking molecules, compounds, or biochemicalconditions according to a change in the T_(m) of a thermal denaturationcurve is preferable. Alternatively, molecules, compounds, or biochemicalconditions can be ranked for their ability to stabilize a targetmolecule according to the change in entire thermal denaturation curve.

[0098] The term “lead molecule” refers to a molecule or compound, from acombinatorial library, which displays relatively high affinity for atarget molecule. The terms “lead compound” and “lead molecule” aresynonymous. The term “relatively high affinity” relates to affinities inthe K_(d) range of from 10⁻⁴ to 10⁻¹⁵ M.

[0099] The term “target molecule” encompasses peptides, proteins,nucleic acids, and other receptors. The term encompasses both enzymesand proteins which are not enzymes. The term encompasses monomeric andmultimeric proteins. Multimeric proteins may be homomeric orheteromeric. The term encompasses nucleic acids comprising at least twonucleotides, such as oligonucleotides. Nucleic acids can besingle-stranded, double-stranded or triple-stranded. The termencompasses a nucleic acid which is a synthetic oligonucleotide, aportion of a recombinant DNA molecule, or a portion of chromosomal DNA.The term target molecule also encompasses portions of peptides,proteins, and other receptors which are capable of acquiring secondary,tertiary, or quaternary structure through folding, coiling or twisting.The target molecule may be substituted with substituents including, butnot limited to, cofactors, coenzymes, prosthetic groups, lipids,oligosaccharides, or phosphate groups. The term “capable of denaturing”refers to the loss of secondary, tertiary, or quaternary structurethrough unfolding, uncoiling, or untwisting. The terms “target molecule”and “receptor” are synonymous.

[0100] Examples of target molecules are included, but not limited tothose disclosed in Faisst, S. et al., Nucleic Acids Research 20:3-26(1992); Pimentel, E., Handbook of Growth Factors, Volumes I-III, CRCPress, (1994); Gilman, A. G. et al., The Pharmacological Basis ofTherapeutics, Pergamon Press (1990); Lewin, B., Genes V, OxfordUniversity Press (1994); Roitt, I., Essential Immunology, BlackwellScientific Publ. (1994); Shimizu, Y., Lymphocyte Adhesion Molecules, R GLandes (1993); Hyams, J. S. et al., Microtubules, Wiley-Liss (1995);Montreuil, J. et al., Glycoproteins, Elsevier (1995); Woolley, P.,Lipases: Their Structure Biochemistry and Applications, CambridgeUniversity Press (1994); Kurjan, J., Signal Transduction: Prokaryoticand Simple Eukaryotic Systems, Academic Press (1993); Kreis, T., et al.,Guide Book to the Extra Cellular Matrix and Adhesion Proteins, OxfordUniversity Press (1993); Schlesinger, M. J., Lipid Modifications ofProteins, CRC Press (1992); Conn, P. M., Receptors: Model Systems andSpecific Receptors, Oxford University Press (1993); Lauffenberger, D. A.et al., Receptors: Models For Binding Trafficking and Signaling, OxfordUniversity Press (1993); Webb, E. C., Enzyme Nomenclature, AcademicPress (1992); Parker, M. G., Nuclear Hormone Receptors; MolecularMechanisms, Cellular Functions Clinical Abnormalities, Academic PressLtd. (1991); Woodgett, J. R., Protein Kinases, Oxford University Press(1995); Balch, W. E. et al., Methods in Enzymology, 257, Pt. C: SmallGTPases and Their Regulators: Proteins Involved in Transport, AcademicPress (1995); The Chaperonins, Academic Press (1996); Pelech, L.,Protein Kinase Circuitry in Cell Cycle Control, R G Landes (1996);Atkinson, Regulatory Proteins of the Complement System, Franklin Press(1992); Cooke, D. T. et al., Transport and Receptor Proteins of PlantMembranes: Molecular Structure and Function, Plenum Press (1992);Schumaker, V. N., Advances in Protein Chemistry: Lipoproteins,Apolipoproteins, and Lipases, Academic Press (1994); Brann, M.,Molecular Biology of G-Protein-Coupled Receptors: Applications ofMolecular Genetics to Pharmacology, Birkhauser (1992); Konig, W.,Peptide and Protein Hormones: Structure, Regulations, Activity—AReference Manual, VCH Publ. (1992); Tuboi, S. et al., Post-TranslationalModification of Proteins, CRC Press (1992); Heilmeyer, L. M., CellularRegulation by Protein Phosphorylation, Springer-Verlag (1991); Takada,Y., Integrin: The Biological Problem, CRC Press (1994); Ludlow, J. W.,Tumor Suppressors: Involvement in Human Disease, Viral ProteinInteractions, and Growth Regulation, R G Landes (1994); Schlesinger, M.J., Lipid Modification of Proteins, CRC Press (1992); Nitsch, R. M.,Alzheimer's Disease: Amyloid Precursor Proteins, Signal Transduction,and Neuronal Transplantation, New York Academy of Sciences (1993);Cochrane, C. G. et al., Cellular and Molecular Mechanisms ofInflammation, Vol. 3: Signal Transduction in Inflammatory Cells, Part A,Academic Press (1992); Gupta, S. et al., Mechanisms of LymphocyteActivation and Immune Regulation IV: Cellular Communications, PlenumPress (1992); Authi, K. S. et al., Mechanisms of Platelet Activation andControl, Plenum Press (1994); Grunicke, H., Signal TransductionMechanisms in Cancer, R G Landes (1995); Latchman, D. S., EukaryoticTranscription Factors, Academic Press (1995).

[0101] The term “target molecule” refers more specifically to proteinsinvolved in the blood coagulation cascade, fibroblast growth factors,and fibroblast growth factor receptors, urokinase, and factor D.

[0102] The term “molecule” refers to the compound which is tested forbinding affinity for the target molecule. This term encompasses chemicalcompounds of any structure, including, but not limited to nucleic acidsand peptides. More specifically, the term “molecule” encompassescompounds in a compound or a combinatorial library. The terms “molecule”and “ligand” are synonymous.

[0103] The terms “thermal change” and “physical change” encompass therelease of energy in the form of light or heat, the absorption of energyin the form or light or heat, changes in turbidity and changes in thepolar properties of light. Specifically, the terms refer to fluorescentemission, fluorescent energy transfer, absorption of ultraviolet orvisible light, changes in the polarization properties of light, changesin the polarization properties of fluorescent emission, changes inturbidity, and changes in enzyme activity. Fluorescence emission can beintrinsic to a protein or can be due to a fluorescence reporter molecule(below). For a nucleic acid, fluorescence can be due to ethidiumbromide, which is an intercalating agent. Alternatively, the nucleicacid can be labeled with a fluorophore (below).

[0104] The term “contacting a target molecule” refers broadly to placingthe target molecule in solution with the molecule to be screened forbinding. Less broadly, contacting refers to the turning, swirling,shaking or vibrating of a solution of the target molecule and themolecule to be screened for binding. More specifically, contactingrefers to the mixing of the target molecule with the molecule to betested for binding. Mixing can be accomplished, for example, by repeateduptake and discharge through a pipette tip. Preferably, contactingrefers to the equilibration of binding between the target molecule andthe molecule to be tested for binding. Contacting can occur in thecontainer (infra) or before the target molecule and the molecule to bescreened are placed in the container.

[0105] The target molecule may be contacted with a nucleic acid prior tobeing contacted with the molecule to be screened for binding. The targetmolecule may be complexed with a peptide prior to being contacted withthe molecule to be screened for binding. The target molecule may bephosphorylated or dephosphorylated prior to being contacted with themolecule to be screened for binding.

[0106] A carbohydrate moiety may be added to the target molecule beforethe target molecule is contacted with the molecule to be screened forbinding. Alternatively, a carbohydrate moiety may be removed from thetarget molecule before the target molecule is contacted with themolecule to be screened for binding.

[0107] The term “container” refers to any vessel or chamber in which thereceptor and molecule to be tested for binding can be placed. The term“container” encompasses reaction tubes (e.g., test tubes, microtubes,vials, etc.). Preferably, the term “container” refers to a well in amultiwell microplate or microtiter plate. The term “sample” refers tothe contents of a container.

[0108] A “thermal denaturation curve” is a plot of the physical changeassociated with the denaturation of a protein or a nucleic acid as afunction of temperature. See, for example, Davidson et al., NatureStructure Biology 2:859 (1995); Clegg, R. M. et al., Proc. Natl. Acad.Sci. U.S.A. 90:2994-2998 (1993).

[0109] The “midpoint temperature, T_(m)” is the temperature midpoint ofa thermal denaturation curve. The T_(m) can be readily determined usingmethods well known to those skilled in the art. See, for example, Weber,P. C. et al., J. Am. Chem. Soc. 116:2717-2724 (1994); Clegg, R. M. etal., Proc. Natl. Acad. Sci. U.S.A. 90:2994-2998 (1993).

[0110] The term “fluorescence probe molecule” refers to a fluorophore,which is a fluorescent molecule or a compound which is capable ofbinding to an unfolded or denatured receptor and, after excitement bylight of a defined wavelength, emits fluorescent energy. The termfluorescence probe molecule encompasses all fluorophores. Morespecifically, for proteins, the term encompasses fluorophores such asthioinosine, and N-ethenoadenosine, formycin, dansyl derivatives,fluorescein derivatives, 6-propionyl-2-dimethylamino)-napthalene(PRODAN), 2-anilinonapthalene, and N-arylamino-naphthalene sulfonatederivatives such as 1-anilinonaphthalene-8-sulfonate (1,8-ANS),2-anilinonaphthalene-6-sulfonate (2,6-ANS),2-aminonaphthalene-6-sulfonate,N,N-dimethyl-2-aminonaphthalene-6-sulfonate,N-phenyl-2-aminonaphthalene,N-cyclohexyl-2-aminonaphthalene-6-sulfonate,N-phenyl-2-aminonaphthalene-6-sulfonate,N-phenyl-N-methyl-2-aminonaph-thalene-6-sulfonate, N-(o-toluyl)-2-aminonaphthalene-6-sulfonate, N-(m-toluyl)-2-aminonaphthalene-6-sulfonate,N-(p-toluyl)-2-aminonaphthalene-6-sulfonate,2-(p-toluidinyl)-naphthalene-6-sulfonic acid (2,6-TNS), 4-(dicyanovinyl)julolidine (DCVJ), 6-dodecanoyl-2-dimethylaminonaphthalene (LAURDAN),6-hexadecanoyl-2-(((2-(trimethylammonium)ethyl)methyl)amino)naphthalenechloride(PATMAN),nile red, N-phenyl-1-naphthylamine,1,1-dicyano-2-[6-(dimethylamino)naphthalen-2-yl]propene (DDNP),4,4′-dianilino-1,1-binaphthyl-5,5-disulfonic acid (bis-ANS), andDapoxyl™ derivatives (Molecular Probes, Eugene, Oreg.). Preferably forproteins, the term refers to 1,8-ANS or 2,6-TNS.

[0111] A double-stranded oligonucleotide may be used in fluorescenceresonance energy transfer assays. One strand of the oligonucleotide willcontain the donor fluorophore. The other strand of the oligonucleotidewill contain the acceptor fluorophore. For a nucleic acid to “contain” adonor or an acceptor fluorophore, the fluorophore can be incorporateddirectly into the oligonucleotide sequence. Alternatively, thefluorophore can be attached to either the 5′- or 3′-terminus of theoligonucleotide.

[0112] A donor fluorophore is one which, when excited by light, willemit fluorescent energy. The energy emitted by the donor fluorophore isabsorbed by the acceptor fluorophore. The term “donor fluorophore”encompasses all fluorophores including, but not limited to,carboxyfluorescein, iodoacetamidofluorescein, and fluoresceinisothiocyanate. The term “acceptor fluorophore” encompasses allfluorophores including, but not limited to, iodoacetamidoeosin andtetramethylrhodamine.

[0113] The term “carrier” encompasses a platform or other object, of anyshape, which itself is capable of supporting at least two containers.The carrier can be made of any material, including, but not limited toglass, plastic, or metal. Preferably, the carrier is a multiwellmicroplate. The terms microplate and microtiter plate are synonymous.The carrier can be removed from the heating element. In the presentinvention, a plurality of carriers are used. Each carrier holds aplurality of containers.

[0114] The term “biochemical conditions” encompasses any component of aphysical, chemical, or biochemical reaction. Specifically, the termrefers to conditions of temperature, pressure, protein concentration,pH, ionic strength, salt concentration, time, electric current,potential difference, concentrations of cofactor, coenzyme, oxidizingagents, reducing agents, detergents, metal ion, ligands, or glycerol.

[0115] The term “denatured protein” refers to a protein which has beentreated to remove secondary, tertiary, or quaternary structure. The term“native protein” refers to a protein which possesses the degree ofsecondary, tertiary or quaternary structure that provides the proteinwith full chemical and biological function. A native protein is onewhich has not been heated and has not been treated with denaturationagents or chemicals such as urea.

[0116] The term “denatured nucleic acid” refers to a nucleic acid whichhas been treated to remove folded, coiled, or twisted structure.Denaturation of a triple-stranded nucleic acid complex is complete whenthe third strand has been removed from the two complementary strands.Denaturation of a double-stranded DNA is complete when the base pairingbetween the two complementary strands has been interrupted and hasresulted in single-stranded DNA molecules that have assumed a randomform. Denaturation of single-stranded RNA is complete whenintramolecular hydrogen bonds have been interrupted and the RNA hasassumed a random, non-hydrogen bonded form.

[0117] The terms “folding,” “refolding,” and “renaturing” refer to theacquisition of the correct secondary, tertiary, or quaternary structure,of a protein or a nucleic acid, which affords the full chemical andbiological function of the biomolecule.

[0118] The term “efficacy” refers to the effectiveness of a particularset of biochemical conditions in facilitating the refolding orrenaturation of an unfolded or denatured protein.

[0119] The terms “spectral measurement” and “spectrophotometricmeasurement” refer to measurements of changes in the absorption oflight. Turbidity measurements, measurements of visible light absorption,and measurement of ultraviolet light absorption are examples of spectralmeasurements.

[0120] The term “polarimetric measurement” relates to measurements ofchanges in the polarization properties of light and fluorescentemission. Circular dichroism and optical rotation are examples ofpolarization properties of light which can be measured polarimetrically.Measurements of circular dichroism and optical rotation are taken usinga spectropolarimeter. “Nonpolarimetric” measurements are those that arenot obtained using a spectropolarimeter.

[0121] The term “collection” refers to a pool or a group of at least onemolecule to be tested for binding to a target molecule or receptor.

[0122] A “host” is a bacterial cell that has been transformed withrecombinant DNA for the purpose of expressing protein which isheterologous to the host bacterial cell.

[0123] The thermal shift assay is based on the ligand-dependent changein the thermal denaturation curve of a receptor, such as a protein or anucleic acid. When heated over a range of temperatures, a receptor willunfold. By plotting the degree of denaturation as a function oftemperature, one obtains a thermal denaturation curve for the receptor.A useful point of reference in the thermal denaturation curve is thetemperature midpoint (T_(m)), the temperature at which the receptor ishalf denatured.

[0124] Ligand binding stabilizes the receptor (Schellman, J.,Biopolymers 14:999-1018 (1975)). The extent of binding and the freeenergy of interaction follow parallel courses as a function of ligandconcentration (Schellman, J., Biophysical Chemistry 45:273-279 (1993);Barcelo, F. et al., Chem. Biol. Interactions 74:315-324 (1990)). As aresult of stabilization by ligand, more energy (heat) is required tounfold the receptor. Thus, ligand binding shifts the thermaldenaturation curve. This property can be exploited to determine whethera ligand binds to a receptor: a change, or “shift”, in the thermaldenaturation curve, and thus in the T_(m), suggests that a ligand bindsto the receptor.

[0125] The thermodynamic basis for the thermal shift assay has beendescribed by Schellman, J. A. (Biopolymers 15:999-1000 (1976)), and alsoby Brandts et al. (Biochemistry 29:6927-6940 (1990)). Differentialscanning calorimetry studies by Brandts et al., (Biochemistry29:6927-6940 (1990)) have shown that for tight binding systems of 1:1stoichiometry, in which there is one unfolding transition, one canestimate the binding affinity at T_(m) from the following expression:$\begin{matrix}{K_{L}^{T_{m}} = \frac{\begin{matrix}{\exp \{ {{- {\frac{\Delta \quad H_{u}^{T_{0}}}{R}\lbrack {\frac{1}{T_{m}} - \frac{1}{T_{0}}} \rbrack}} +} } \\ \quad {\frac{\Delta \quad C_{pu}}{R}\lbrack {{\ln ( \frac{T_{m}}{T_{0}} )} + \frac{T_{0}}{T_{m}} - 1} \rbrack} \}\end{matrix}}{\lbrack L_{T_{m}} \rbrack}} & ( {{equation}\quad 1} )\end{matrix}$

[0126] where K_(L) ^(T) ^(_(m)) =the ligand association constant atT_(m);

[0127] T_(m)=the midpoint for the protein unfolding transition in thepresence of ligand;

[0128] T₀=the midpoint for the unfolding transition in the absence ofligand;

[0129] ΔH_(u) ^(T) ^(₀) =the enthalpy of protein unfolding in theabsence of ligand at T₀;

[0130] ΔC_(pu)=the change in heat capacity upon protein unfolding in theabsence of ligand;

[0131] [L_(T) _(m) ]=the free ligand concentration at T_(m); and

[0132] R=the gas constant.

[0133] The parameters ΔH_(u) and ΔC_(pu) are usually observed fromdifferential scanning calorimetry experiments and are specific for eachreceptor. To calculate the binding constant from equation 1, one shouldhave access to a differential scanning calorimetry instrument to measureΔH_(u) and ΔC_(pu) for the receptor of interest. One can also locatethese parameters for the receptor of interest, or a receptor closelyrelated to it, in the literature. In these situations, equation (1) willallow the accurate measurement of K_(L) at T_(m).

[0134] It is also possible to calculate the ligand association constantat any temperature, K_(L) at T, using equation 2. To use equation 2,calorimetry data for the binding enthalpy at T, ΔH_(L), and the changeof heat capacity upon ligand binding, ΔC_(pL) must be known (Brandts etal., Biochemistry 29:6927-6940 (1990)). $\begin{matrix}{K_{L}^{T} = {K_{L}^{T_{m}}\exp \{ {{- {\frac{\Delta \quad H_{L}^{T}}{R}\lbrack {\frac{1}{T} - \frac{1}{T_{m}}} \rbrack}} + {\frac{\Delta \quad C_{pL}}{R}\lbrack {{\ln ( \frac{T}{T_{m}} )} - \frac{T}{T_{m}} + 1} \rbrack}} \}}} & ( {{equation}\quad 2} )\end{matrix}$

[0135] where K_(L) ^(T)=the ligand association constant at anytemperature T;

[0136] K_(L) ^(T) ^(_(m)) =the ligand association constant at T_(m);

[0137] T_(m)=the midpoint for the protein unfolding transition in thepresence of ligand;

[0138] ΔH_(L) ^(T)=the enthalpy of ligand binding in the absence ofligand at T;

[0139] ΔC_(pL)=the change in heat capacity upon binding of ligand; and

[0140] R=the gas constant.

[0141] The second exponential term of equation 2 is usually small enoughto be ignored so that approximate values of K_(L) at T can be obtainedusing just the first exponential term: $\begin{matrix}{K_{L}^{T} = {K_{L}^{T_{m}}\exp \{ {- {\frac{\Delta \quad H_{L}^{T}}{R}\lbrack {\frac{1}{T} - \frac{1}{T_{m}}} \rbrack}} \}}} & ( {{equation}\quad 3} )\end{matrix}$

[0142] One need not, however, calculate binding constants according toequations 1-3 in order to rank the affinities of a multiplicity ofdifferent ligands for a receptor. Rather, the present invention providesa method for ranking affinities of ligands according to the degree towhich the thermal denaturation curve is shifted by the ligand. Thus, itis possible to obtain estimates of at T_(m), even in the absence ofaccurate values of ΔH_(u), ΔC_(pu), and ΔH_(L).

[0143] The present invention is particularly useful for screening acombinatorial or a compound library. To achieve high throughputscreening, it is best to house samples on a multicontainer carrier orplatform. A multicontainer carrier facilitates the heating of aplurality of samples simultaneously. In one embodiment, a multiwellmicroplate, for example a 96 or a 384 well microplate, which canaccommodate 96 or 384 different samples, is used as the earner.

[0144] In one embodiment, one sample is contained in each well of amulti-well microplate. The control well contains receptor, but nomolecule to be tested for binding. Each of the other samples contains atleast one molecule to be tested for binding. The thermal denaturationcurve for the receptor in the control well is the curve against whichcurves for all of the other experiments are compared.

[0145] The rate of screening is accelerated when the sample containsmore than one molecule to be tested for binding. For example, thescreening rate is increased 20-fold when the sample contains a pool of20 molecules. Samples which contain a binding molecule must then bedivided into samples containing a smaller collection of molecules to betested for binding. These divided collections must then be assayed forbinding to the target molecule. These steps must be repeated until asingle molecule responsible for the original thermal shift is obtained.

[0146] Receptor denaturation can be measured by light spectrophotometry.When a protein in solution denatures in response to heating, thereceptor molecules aggregate and the solution becomes turbid. Thermallyinduced aggregation upon denaturation is the rule rather than theexception. Aggregation generally complicates calorimetric experiments.Aggregation, however, is an advantage when, using a spectrophotometrictechnology, because changes in turbidity can be measured by monitoringthe change in absorbance of visible or ultraviolet light of a definedwavelength.

[0147] Denaturation of a nucleic acid can be monitored using lightspectrophotometry. The change in hyperchromicity, which is the increasein absorption of light by polynucleotide solutions due to a loss ofordered structure, is monitored as a function of increase intemperature. Changes in hyperchromicity is typically assayed using lightspectrophotometry.

[0148] In another embodiment, however, fluorescence spectrometry is usedto monitor thermal denaturation. The fluorescence methodology is moresensitive than the absorption methodology.

[0149] The use of intrinsic protein fluorescence and fluorescence probemolecules in fluorescence spectroscopy experiments is well known tothose skilled in the art. See, for example, Bashford, C. L. et al.,Spectrophotometry and Spectrofluorometry: A Practical Approach, pp.91-114, IRL Press Ltd. (1987); Bell, J. E., Spectroscopy inBiochemistry, Vol. 1, pp. 155-194, CRC Press (1981); Brands L. et al.,Ann. Rev. Biochem. 41:843 (1972).

[0150] If the target molecule or receptor to be studied is a nucleicacid, fluorescence spectrometry can be performed using an ethidiumbromide displacement assay (Lee, M. et al., J. Med. Chem. 36:863-870(1993)). In this approach, ligand binding displaces ethidium bromide andresults in a decrease in the fluorescent emission from ethidium bromide.An alternative approach is to use fluorescence resonance emissiontransfer. In the latter approach, the transfer of fluorescent energy,from a donor fluorophore on one strand of an oligonucleotide to anacceptor fluorophore on the other strand, is monitored by measuring theemission of the acceptor fluorophore. Denaturation prevents the transferof fluorescent energy.

[0151] The fluorescence resonance emission transfer methodology is wellknown to those skilled in the art. Fore example, see Ozaki, H. et al.,Nucleic Acids Res. 20:5205-5214 (1992); Clegg, R. M. et al., Proc. Natl.Acad. Sci. U.S.A. 90:2994-2998 (1993); Clegg, R. M. et al., Biochemistry31:4846-4856 (1993).

[0152] The element upon which the sample carrier is heated can be anyelement capable of heating samples rapidly and in a reproduciblefashion. In the present invention, a plurality of samples is heatedsimultaneously. The plurality of samples can be heated on a singleheating element. Alternatively, the plurality of samples can be heatedto a given temperature on one heating element, and then moved to anotherheating element for heating to another temperature. Heating can beaccomplished in regular or irregular intervals. To generate a smoothdenaturation curve, the samples should be heated evenly, in intervals of1 or 2° C. The temperature range across which the samples can be heatedis from 25 to 110° C. Spectral readings are taken after each heatingstep. Samples can be heated and read by the spectral device in acontinuous fashion: Alternatively, after each heating step, the samplesmay be cooled to a lower temperature prior to taking the spectralreadings. Preferably, the samples are heated continuously and spectralreadings are taken while the samples are being heated.

[0153] Spectral readings can be taken on all of the samples in thecarrier simultaneously. Alternatively, readings can be taken on samplesin groups of at least two at a time. Finally, the readings can be takenone sample at a time.

[0154] In one embodiment, thermal denaturation is monitored byfluorescence spectrometry using an assay apparatus such as the one shownin FIG. 29. The instrument consists of a scanner and a control softwaresystem. The system is capable of quantifying soluble and cell-associatedfluorescence emission. Fluorescence emission is detected by aphotomultiplier tube in a light-proof detection chamber. The softwareruns on a personal computer and the action of the scanner is controlledthrough the software. A precision X-Y mechanism scans the microplatewith a sensitive fiber-optic probe to quantify the fluorescence in eachwell. The microplate and samples can remain stationary during thescanning of each row of the samples, and the fiber-optic probe is thenis moved to the next row. Alternatively, the microplate and samples canbe moved to position a new row of samples under the fiber-optic probe.The scanning system is capable of scanning 96 samples in under oneminute. The scanner is capable of holding a plurality of excitationfilters and a plurality of emission filters to measure the most commonfluorophores. Thus, fluorescence emission readings can be taken onesample at a time, or on a subset of samples simultaneously. An alternateembodiment of the assay apparatus is shown in FIG. 33. The assayapparatus of the present invention will be described in more detailbelow.

[0155] The present invention is also directed to a product ofmanufacture which comprises a carrier having a multiplicity ofcontainers within it. The product of manufacture can be used to screen acombinatorial library for lead compounds which bind to the receptor ofinterest The combinatorial library can be screened using the methodaccording to the present invention.

[0156] In the product of manufacture, each of the containers contains auniform amount of a receptor of interest In addition, each of thesecontainers contains a different compound from a combinatorial library ata concentration which is at least 2-fold above the concentration of thereceptor. Preferably, the product of manufacture is a multiwellmicroplate or a multiplicity of multiwell microplates. If the receptoris a protein, each container may further contain a fluorescence probemolecule. If the receptor is a nucleic acid, each container may furthercontain ethidium bromide. Alternatively, the nucleic acid may be labeledwith a fluorophore.

[0157] Prior to use, the product of manufacture can be stored in anymanner necessary to maintain the integrity of the receptor of interest.For example, the product of manufacture can be stored at a temperaturebetween −90° C. and room temperature. The receptor and compound can bestored in lyophilized form, in liquid form, in powdered form, or can bestored in glycerol. The product of manufacture may be stored either inthe light or in the dark.

[0158] The heat conducting element or block upon which the samplecarrier is heated can be any element capable of heating samples rapidlyand reproducibly. The plurality of samples can be heated on a singleheating element. Alternatively, the plurality of samples can be heatedto a given temperature on one heating element, and then moved to anotherheating element for heating to another temperature. Heating can beaccomplished in regular or irregular intervals. To generate a smoothdenaturation curve, the samples should be heated evenly, in intervals of1 or 2° C. The temperature range across which the samples can be heatedis from 25 to 110° C.

[0159] In the present invention, a plurality of samples is heatedsimultaneously. If samples are heated in discrete temperature intervals,in a stairstep fashion, spectral readings are taken after each heatingstep. Alternatively, after each heating step, the samples may be cooledto a lower temperature prior to taking the spectral readings.Alternatively, samples can be heated in a continuous fashion andspectral readings are taken during heating.

[0160] Spectral readings can be taken on all of the samples on a carriersimultaneously. Alternatively, readings can be taken on samples ingroups of at least two at a time.

[0161] The present invention also provides an improved method forgenerating lead compounds. After a compound or a combinatorial libraryof compounds has been screened using the thermal shift assay, compoundswhich bind to the target receptor are chemically modified to generate asecond library of compounds. This second library is then screened usingthe thermal shift assay. This process of screening and generating a newlibrary continues until compounds that bind to the target receptor withaffinities in the K_(d) range of from 10⁻⁴ to 10⁻¹⁵ M are obtained.

[0162] A fluorescence emission imaging system can be used to monitor thethermal denaturation of a target molecule or a receptor. Fluorescenceemission imaging systems are well known to those skilled in the art. Forexample, the AlphaImager™ Gel Documentation and Analysis System (AlphaInnotech, San Leandro, Calif.) employs a high performancd charge coupleddevice camera with 768×494 pixel resolution. The charge coupled devicecamera is interfaced with a computer and images are anlayzed with Imageanalysis software™. The ChemiImager™ (Alpha Innotech) is a cooled chargecoupled device that performs all of the functions of the AlphaImager™and in addition captures images of chemiluminescent samples and otherlow intensity samples. The ChemiImager™ charge coupled device includes aPentium processor (1.2 Gb hard drive, 16 Mb RAM), AlphaEase™ analysissoftware, a light tight cabinet, and a UV and white lighttrans-illuminator. For example, the MRC-1024 UV/Visible Laser ConfocalImaging System (BioRad, Richmond, Calif.) facilitates the simultaneousimaging of more than one fluorophore across a wide range of illuminationwavelengths (350 to 700 nm). The Gel Doc 1000 Fluorescent GelDocumentation System (BioRad, Richmond, Calif.) can clearly displaysample areas as large as 20×20 cm, or as small as 5×4 cm. At least two96 well microplates can fit into a 20×20 cm area. The Gel Doc 1000system also facilitates the performance of time-based experiments.

[0163] A fluorescence thermal imaging system can be used to monitorreceptor unfolding in a microplate thermal shift assay. In thisembodiment, a plurality of samples is heated simultaneously between 25to 110° C. A fluorescence emission reading is taken for each of theplurality of samples simultaneously. For example, the fluorescenceemission in each well of a 96 or a 384 well microplate can be monitoredsimultaneously. Alternatively, fluorescence emission readings can betaken continuously and simultaneously for each sample. At lowertemperatures, all samples display a low level of fluorescence emission.As the temperature is increased, the fluorescence in each sampleincreases. Wells which contain ligands which bind to the target moleculewith high affinity shift the thermal denaturation curve to highertemperatures. As a result, wells which contain ligands which bind to thetarget molecule with high affinity fluoresce less, at a giventemperature above the T_(m) of the target molecule in the absence of anyligands, than wells which do not contain high-affinity ligands. If thesamples are heated in incremental steps, the flourescence of all of theplurality of samples is simultaneoulsy imaged at each heating step. Ifthe samples are heated continuously, the fluorescent emission of all ofthe plurality of samples is simultaneously imaged during heating.

[0164] A thermal shift assay can be performed in a volume of 100 μLvolumes. For the following reasons, however, it is preferable to performa thermal shift assay in a volume of 10 μL. First, approximately 10-foldless protein is required for the miniaturized assay. Thus, only ˜5 to 40pmole of protein are required (0.1 μg to 1.0 μg for a 25 kDa protein)for the assay (i.e. 5 to 10 μL working volume with a target moleculeconcentration of about 1 to about 4 μM). Thus, 1 mg of protein can beused to conduct 1,000 to 10,000 assays in the miniaturized format Thisis particularly advantageous when the target molecule is available inminute quantities.

[0165] Second, approximately 10-fold less ligand is required for theminiaturized assay. This advantage is very important to researchers whenscreening valuable combinatorial libraries for which library compoundsare synthesized in minute quantities. In the case of human α-thrombin,the ideal ligand concentration is about 50 μM, which translates into 250pmoles of ligand, or 100 ng (assuming a MW of 500 Da) of ligand perassay in the miniaturized format.

[0166] Third, the smaller working volume allows the potential of usinglarger arrays of assays because the miniaturized assay can fit into amuch smaller area. For example, a 384 well (16×24 array) or 864 well(24×36 array) plates have roughly the same dimensions as the 96 wellplates (about 8.5×12.5 cm). The 384 well plate and the 864 well plateallows the user to perform 4 and 9 times as many assays, respectively,as can be performed using a 96 well plate. Alternatively, 1536 wellplates (32×48 arrays; Matrix Technologies Corp.) can be used. A 1536well plate will facilitate sixteen times the throughput afforded by a 96well plate.

[0167] Thus, using the 1536 well plate configuration, the assay speedcan be increased by about 16 times, relative to the speed at which theassay can be performed using the 96 well format. The 8×12 assay arrayarrangement (in a 96-well plate) facilitates the performance of 96assays/hr, or about 2300 assays/24 hours. The 32×48 array assayarrangement facilitates the performance of about 1536 assays hr., orabout 37,000 assays/24 hours can be performed using a 32×48 assay arrayconfiguration.

[0168] The assay volume can be 1-100 μL. Preferably, the assay volume is1-50 μL. More preferably, the assay volume is 1-25 μL. More preferablystill, the assay volume is 1-10 μL. More preferably still, the assayvolume is 1-5 μL. More preferably still, the assay volume is 5 μL. Mostpreferably, the assay volume is 1 μL or 2 μL.

[0169] Preferably, the assay is performed in V-bottom polycarbonateplates or polycarbonate dimple plates. A dimple plate is a plate thatcontains a plurality of round-bottom wells that hold a total volume of15 μL.

[0170] One alternative to taking spectral readings over a temperaturerange around the T_(m) of the therapeutic target to obtain a fullthermal unfolding curve for the ligand/target complex, in order toidentify shifts in T_(m), is to perform the assay at a singletemperature near the T_(m) of the target molecule. In this embodiment,samples that emit less fluorescence, relative to a control sample(containing a target molecule, but no candidate ligand) indicate thatthe candidate ligand binds to the target molecule.

[0171] In this embodiment, the magnitude of a physical change associatedwith the thermal denaturation of a target molecule resulting fromheating is determined by generating a thermal denaturation curve for thetarget molecule as a function of temperature over a range of one or morediscrete or fixed temperatures. The physical change associated withthermal denaturation, for example, fluorescence emission, is measured.The magnitude of the physical change at the discrete or fixedtemperature for the target molecule in the absence of any ligand isnoted. The magnitude of the physical change in the presence of each of amultiplicity of different molecules, for example, combinatorialcompounds, is measured. The magnitude of the physical change associatedwith thermal denaturation of the target molecule in the presence of eachof the multiplicity of molecules is compared to magnitude of thephysical change obtained for the target molecule at the discrete orfixed temperature in the absence of any of the multiplicity of differentmolecules. The affinities of the multiplicity of different molecules areranked according to the change in the magnitude of the physical change.

[0172] The discrete or fixed temperature at which the physical change ismeasure can be any temperature that is useful for discriminating shiftsin thermal stability. Preferably, the discrete or fixed temperature isthe midpoint temperature T_(m) for the thermal denaturation curve forthe target molecule in the absence of any of the multiplicity ofdifferent molecules tested for binding to the target molecule.

[0173] The single temperature configuration is particularly advantageousif one is interested in assaying a series of relatively high affinityligands, which are the preferred compounds for candidates in clinicaltesting. In cases where a less stringent requirement for bindingaffinity is preferred, however, one may increase the ligandconcentration to 500 μM in order to identify ligands with K_(d)'s of 2.5μM or higher affinity.

[0174] The single temperature embodiment offers a number of advantages.First, assay speed is increased by a factor often fold. Thus, as the 96well plate (8×12 array) assay facilitates about 96 assays per hour, thesingle temperature variation will facilitate about 1000 assays per hr.Using a 1536 well plate (32×48 array), as long as sample aliquoting canbe effected at the same rate for the 32×48 array system as in the 8×12array system, about 15,000 assays can be performed per hour.

[0175] Another alternative method for detecting the thermal unfoldingtransitions for the microplate thermal shift assay is through theintrinsic tryptophan (Trp) fluorescence of the target protein. Mostfluorescence emission plate readers, such as the CytoFluor II, usetungsten-halogen lamps as their light source. These lamps do not giveoff enough light near 280 nm to allow excitation of the intrinsic Trpresidues of proteins which absorb maximally near 280 nm. However, theBiolumin 960 (Molecular Dynamics) uses a Xenon-Arc lamp. The Xenon-Arclamp affords excitation at 280 nm and the measurement of emission at 350nm.

[0176] The methods and assay apparatus of the present invention are notlimited to assaying ligand-protein interactions. The methods and theassay apparatus can be used to rapidly assay any multi-variable systemrelated to protein stabilization. For example, the methods and the assayapparatus of the present invention can be used to simultaneously assaythe binding of more than one compound or ligand to a target molecule.Using this approach, the additive effect of multiple-ligand binding canbe assessed. Positive and negative cooperativity can be determined. Toaccomplish this method, thermal shift assays are performed for a targetmolecule, such as a protein, in the absence of any ligands, in thepresence of a single ligand, and in the presence of two or more ligands.A thermal denaturation curve is generated for the protein alone and foreach combination of protein and ligand(s). The midpoint temperatureT_(m) is then determined for each curve. Each T_(m) is then compared toeach of the other T_(m)'s for the other curves. Alternatively, eachentire thermal denaturation curve is compared to each of the otherthermal denaturation curves. In either of these manners, the additivecontribution of more than one ligand to binding interaction or toprotein stability can be determined.

[0177] In a similar fashion, the additive contributions of one or morebiochemical conditions to protein stability can be determined. Thus, thepresent invention can be used to rapidly identify biochemical conditionsthat optimize protein stabililty, and hence shelf-life. of a protein.

[0178] Further, the methods and the assay apparatus of the presentinvention can be used to rank the efficacies of various biochemicalconditions for refolding or renaturing an unfolded or denatured protein.This embodiment addresses the need in the art for a reliable method forscreening for effective refolding or renaturing conditions.

[0179] For example, expression of recombinant DNA in a bacterial cellusually results in the sequestration of recombinant protein intobacterial inclusion bodies (Marston, F. A. O., Biochem. J. 240:1-12(1986)). Although other expression systems can be used instead ofbacterial expression systems, expression in bacterial cells remains themethod of choice for the high-level production of recombinant proteins(Rudolph, R., Protein Engineering: Principles and Practices, pp.283-298, John Wiley & Sons (1995)). In many cases, recovery ofrecombinant protein requires that protein be isolated from inclusionbodies. Protein purification from inclusion bodies process necessitatesthe denaturation of recombinant protein. As a result, recombinantprotein must be renatured or refolded under conditions suitable togenerate the protein in its native, fully functional form.

[0180] In each of these cases, denatured protein must be renatured orrefolded in order to be useful for further study or use. Unfortunately,one cannot easily predict the exact conditions under which a givenprotein or fragment of the protein should be renatured. Each protein isdifferent. One must always resort to testing a number of differentcombinations of renaturing conditions before one can know which set ofconditions is optimal. Thus, it is desirable to have a reliable andrapid method for ranking the efficacies of various renaturingconditions.

[0181] Recombinant DNA technology has allowed the biosynthesis of a widevariety of heterologous polypeptides of interest in relatively largequantities through the recruitment of the bacterial protein expressionapparatus. However, the promise of cheap and abundant supplies ofcorrectly folded rare human proteins of high therapeutic value expressedin E. coli has foundered due to the overwhelmingly predominantaggregation of unfolded or partially unfolded target proteins intoinsoluble protein inclusion bodies., For recent reviews, see Rudolph,R., & Lilie, H., FASEB J. 10:49-56 (1995); Sadana, A., Biotechnology &Bioengineering 48:481-489 (1995); Jaenicke, R., Phil. Trans. Royal Soc.London Ser. B-Biol. Sci. 348:97-105 (1995)). Reasons for the prevailingself aggregation reaction in E. coli have centered on the relativelyhigh concentration of the heterologous protein (as high as 30% of theweight of the cell) found to various degrees in partially unfoldedstates. Thus, at the elevated protein concentrations of anoverexpressing E. coli stain, the exposed hydrophobic residues ofunfolded proteins are more likely to encounter other molecules withsimilarly exposed groups (inter-molecular reaction) than they are tosample self collapsed polypeptide conformations where these hydrophobicresidues are packed in a proper orientation (intra-molecular transitionstate) for proceeding to the fully folded native state (see FIG. 26).From this perspective, the insoluble protein inclusion bodies are seenas kinetically trapped side reaction products that thwart the preferredprotein folding process.

[0182] Techniques for isolating inclusion bodies, purifying recombinantprotein from inclusion bodies, and techniques for refolding orrenaturing protein are well known to those skilled in the art Forexample, see Sambrook, J. et al., Molecular Cloning: a LaboratoryManual, pp. 17.37-17.41, Cold Spring Harbor Laboratory Press (1989);Rudolph, R. et al., FASEB J. 10:49-56 (1995).

[0183] Another impediment to producing large quantities of correctlyfolded proteins in E. coli is that the reducing redox potential of theE. coli cytosol impedes the formation of disulfide bonds in vivo. Theformation of disulfide bonds is an important co- and post-translationalevent in the biosynthesis of many extracellular proteins that is oftencoupled to protein folding. In addition, the cis-trans prolineisomerization reaction has been demonstrated to be a rate determiningstep for correct folding of certain proteins (Lin, L.-N., & Brandts, J.F., Biochemistry 22:564-573 (1983)). As a result, partially foldedintermediates accumulate in sufficient quantity in vivo that theyaggregate and precipitate into protein masses.

[0184] Cells employ a class of host proteins called molecularchaperonins that assist in vivo protein folding by apparently preventingmany of the unproductive side reactions discussed above with regard toinclusion body formation, i.e. aggregation and improper disulfide bondformation. However, the E. coli chaperonin machinery, which is comprisedin part by the proteins, GroEL and GroES, presumably becomes overwhelmedby massive overexpression. Despite many attempts to correct thischaperonin deficit by co-expression of molecular chaperonins with theprotein of interest (Rudolph, R., & Lilie, H., The FASEB J. 10:49-56(1995)) positive results have been reported in only one case(Goloubinoff, P., et al., Nature 342:884-889 (1989)).

[0185] Two hypotheses have been promoted to explain how GroEL and GroESassist in vivo protein folding. Under the first hypothesis, the Anfinsencage hypothesis, the function of a molecular chaperonin is to provide aprotected environment where folding of the protein to its native statecan proceed without interference by pro-aggregation conditions in thecell (Martin, et al., Nature 352:36-42 (1991); Ellis, R. J., CurrentBiology 4:633-635 (1994)). Under the second hypothesis, the “iterativeannealing” hypothesis, the function of the chaperonin is to partlyunfold misfolded proteins (that is, kinetically trapped intermediates)with some of the energy of ATP hydrolysis being channeled into theconformational energy of the substrate polypeptide, forcing thepolypeptide into a higher energy state from which it could once againattempt to refold correctly after being released into solution (Todd, M.J. et al., Science 265:659-666 (1994); Jackson, et al., Biochemistry32:2554-2563 (1993); Weissman, J. S., et al., Cell 78:693-702 (1994);Weissman, J. S., & Kim, P. S., Science 253:1386-1393 (1991)).

[0186] The in vivo results discussed above are in many ways consistentwith the more recent experiences with in vitro refolding of recombinantheterologous proteins expressed in E. coli. That is, while the primaryamino acid sequence of a protein may contain sufficient information todetermine its native folded conformation (Anfinsen, C. B., Science181:223-230 (1973)), the biochemical conditions in which the foldingreaction takes place can strongly influence the partitioning betweenunfolded, aggregated, and correctly folded forms.

[0187] For example, pH can be understood to influence the foldingreaction by its effect on the long range electrostatic interactionssummed in the fourth term of the equation (4).

ΔG _(fold) =ΔG _(conf) +ΣΔg _(i,int) +ΣΔg _(i,s) +ΔW _(el)+(ΔG_(bind))  Equation (4)

[0188] where ΔG_(conf)=conformational free energy (order/disorder term);

[0189] Δg_(i,int)=short range interactions (H-bonds, van der Wallsinteractions, salt bridges, cofactor binding, etc.);

[0190] Δg_(i,s)=short range interactions with solvent (hydrophobiceffect, hydration of ions, etc.); and

[0191] ΔW_(el)=long range electrostatic interactions.

[0192] ΔG_(bind)=ligand binding free energy

[0193] As the pH of a protein solution is lowered below the pI for theprotein, functional groups on the polypeptide become increasinglyprotonated, to the point where the electrostatic repulsion betweenfunctional groups eventually out balances the other terms in the freeenergy equation (equation (4)), and the protein is no longer able toadopt the native conformation.

[0194] Another important biochemical parameter for protein folding isthe solvent, water, which repels aliphatic and aromatic side chains (andpossibly the main chain to some extent) to minimize their exposedsurface area. The influence of solvent over the folding reaction issummed in the third term of the free energy equation (equation (4)).Certain salts are known to increase the hydrophobic interaction amongprotein side chains in water solutions. The effect depends upon thenature of the ions following the Hofmeister series: Cations:Mg²⁺>Li⁺>Na+>K⁺>NH₄ ⁺. Anions: SO₄ ²⁻>HPO₄²⁻>acetate>citrate>tartrate>Cl⁻>NO₃−>ClO₃ ⁻>I⁻>ClO₄ ⁻>SCN⁻. StabilizingHofmeister anions, such as SO₄ ²⁻ and HPO₄ ²⁻ at 0.4 M have been foundto increase the yield of correctly folded proteins (Creighton, T. E.,In: Proteins: Structures and Molecular Properties, Freeman, New York,(1984)). This favorable outcome for the native conformation of theprotein has been attributed to the cations' and anions' “salting out”effect which leads to the preferential hydration of the protein(Creighton, T. E., In: Proteins: Structures and Molecular Properties,Freeman, New York, (1984)).

[0195] Glycerol alters the solvation properties of water to favor thenative conformation of proteins. The mechanism by which this occurs isthe co-solvent exclusion and preferential hydration of the protein, notunlike the effect of salts of the salts of the Hofmeister series(Timasheff & Arakawa, In: Protein Structure, A Practical Approach, T. E.Creighton, ed., IRL Press, Oxford, UK (1989), pp. 331-354).

[0196] Another example of how the environment influences protein foldingis the effect that known ligands and cofactors have on the yield offolded protein. Ligand binding has the effect of shifting theequilibrium from an unfolded state to a native-ligand complex through acoupling of the binding free energy to that of the folding reaction. Therole of metal ions in the refolding of bovine carbonic anhydrase II hasbeen described (Bergenhem & Carlsson, Biochim. Biophys. Acta 998:277-285(1989)). Other biochemical parameters that have been shown to affectprotein folding are: protein concentration, temperature, glutathioneredox buffers (GSH, GSSG), the presence of detergents, and the presenceof other additives, such as glycerol, arginine-HCl, polyethylene glycol(PEG), and organic solvents.

[0197] During incubation under refolding conditions, recombinantproteins can be immobilized to solid phase support. This configurationresembles the “Anfinsen cage” hypothesis for the function of GroEL andGroES where an unfolded protein becomes temporarily immobilized in aprotected environment where folding to the native state can proceedwithout interference from competing aggregation reactions. Confirmationof protein folding on solid supports has now come from two recentreports in the literature. A poly-histidine tagged TIMP-2 protein couldbe refolded by dialysis while still bound to a metal chelate column(Negro, A. et al., FEBS Lett. 360:52-56 (1995)). A polyionic fusionpeptide attached to the amino or carboxyl terminus of α-glucosidaseallowed folding while bound to heparin-Sepharose resin at about 5 mg/mL(Rudolph & Lilie, FASEB J. 10:49-56 (1995)). A polyionic arginine tagmetholdology for immobilizing and refolding a-glucosidase is disclosedin Stempfer, G. et al., Nature Biotechnology 14:329-334 (1996).

[0198] In the present invention, the thermal shift assay is used to rankthe efficacy of various refolding or renaturing conditions. Each of amultiplicity of aliquots of a protein of interest, which has beenincubated under a variety of different biochemical folding conditions,are placed in a container in a multicontainer carrier. An aliquot of thenative, fully functional protein of known concentration is placed in thecontrol container. The samples can be placed in any multicontainercarrier. Preferably, each sample can be placed in a well of a multiwellmicroplate.

[0199] In considering the many biochemical variables that can influencethe outcome of the protein folding reaction, optimization of proteinfolding is a multi-variable optimization problem, not unlike proteincrystallization and quantitative structure activity relationships (QSAR)in drug discovery. Multi-variable optimization problems require largenumbers of parallel experiments to collect as much data as possible inorder to influence a favorable response. In this regard, both proteincrystallization and QSAR analyses have greatly benefited from massscreening protocols that employ matrix arrays of incremental changes inbiochemical or chemical composition.

[0200] The present invention can be used to rank the efficacies ofrefolding or renaturing conditions. Such conditions include, but are notlimited to, the concentration of glycerol, the concentration of protein,the use of agents which catalyze the formation of disulfide bondformation, temperature, pH, ionic strength, type of solvent, the use ofthiols such as reduced glutathione (GSH) and oxidized glutathione(GSSG), chaotropes such as urea, guanidinium chlorides, alkyl-urea,organic solvents such as carbonic acid amides, L-arginine HCl, Trisbuffer, polyethylene glycol, nonionic detergents, ionic detergents,zwitterionic detergents, mixed micelles, and a detergent in combinationwith cyclodextrin. The present invention can be used regardless ofwhether a denaturation agent is removed from the protein using dialysis,column chromatographic techniques, or suction filtration.

[0201] Using a spectral thermal shift assay, the conditions whichfacilitate optimal protein refolding can be determined rapidly. In thisembodiment, the renatured protein samples and a control protein sample(i.e., a sample of native protein in its fully functional form) areheated over a temperature range. At discrete temperature intervals, aspectral reading is taken. Alternatively, spectral readings can be takenduring a continuous, pre-determined temperature profile. A thermaldenaturation curve is constructed for each sample. The T_(m) for thethermal denaturation curve of the native, fully functional protein isdetermined. The relative efficacies of the refolding conditions areranked according to the magnitude of the physical change associated withunfolding at the T_(m) of the native, fully functional protein, relativeto the magnitude of the physical change of a known quantity of thenative, fully functional protein at that T_(m). The magnitude ofphysical change used to measure the extent of unfolding (reflected onthe ordinate, or y-axis, of a thermal denaturation curve) corresponds tothe amount of correctly folded protein.

[0202] The present invention provides a method for screening biochemicalconditions that facilitate and optimize protein folding. To screenconditions for a given protein, it is first necessary to determine thethermal unfolding profile for a protein of interest. This isaccomplished by generating a denaturation curve using the microplatethermal shift assay. Various conditions can be optimized, including pHoptimum, ionic strength dependence, concentration of salts of theHofmeister series, glycerol concentration, sucrose concentration,arginine concentration, dithiothreitol concentration, metal ionconcentration, etc.

[0203] Using the microplate thermal shift assay, one can determine oneor more biochemical conditions have an additive effect on proteinstability. Once a set of biochemical conditions that facilitate anincrease in protein stability have been identified using the thermalshift assay, the same set of conditions can be used in protein foldingexperiments with recombinant protein. See FIG. 27. If the conditionsthat promote protein stability in the thermal shift assay correlate withconditions that promote folding of recombinant protein, conditions canbe further optimized by performing additional thermal shift assays untila combination of stabilizing conditions that result in further increaseprotein stability are identified. Recombinant protein is then foldedunder those conditions. This process is repeated until optimal foldingconditions are identified. Protein stability is expected to correlatewith improved yields of protein folding. Yield of correctly foldedprotein can be determined using any suitable technique. For example,yield of correctly folded protein can be calculated by passing refoldedprotein over an affinity column, for example, a column to which a ligandof the protein is attached, and quantifying the amount of protein thatis present in the sample. In this way, folding conditions can beassessed for their additive contributions to correct folding. Thetransition state for the protein folding reaction resembles the nativeform of the protein more than the denatured form. This has beendemonstrated to be the case for may proteins (Fersht, A. R., Curr. Op.Struct. Biol. 7:3-9 (1997)).

[0204] The methods and the apparatus of the present invention provide arapid, high throughput approach to screening for combinations ofbiochemical conditions that favor the protein folding. The method doesnot require cumbersome and time consuming steps that conventionalapproaches to protein folding require. For example, using the method ofthe present invention, it is not necessary to dilute protein to largevolumes and low protein concentrations (˜10 μg/mL) in order to avoidaggregation problems associated with conventional methods of recombinantprotein refolding. Suppression of protein aggregation will allow forscreening biochemical parameters that shift the protein foldingequilibrium (between the unfolded and the folded forms of proteins) tothe correct native conformation.

[0205] Like protein stabilization, protein folding, ligand selection,and drug design, selection of conditions that promote proteincrystallization is another multi-variable optimization problem that issolved using the methods and the apparatus of the present invention.

[0206] The methods and the assay apparatus of the present invention arealso useful for determining conditions that facilitate proteincrystallization. The crystallization of molecules from solution is areversible equilibrium process, and the kinetic and thermodynamicparameters are a function of the chemical and physical properties of thesolvent system and solute of interest (McPherson, A., In: Preparationand Analysis of Protein Crystals, Wiley Interscience (1982); Weber, P.C., Adv. Protein Chem. 41:1-36 (1991)) 1991). Under supersaturatingconditions, the system is driven toward equilibrium where the solute ispartitioned between the soluble and solid phase instead of the unfoldedand native states. The molecules in the crystalline phase pack inordered and periodic three dimensional arrays that are energeticallydominated by many of the same types of cohesive forces that areimportant for protein folding, i.e. van der Waals interactions,electrostatic interactions, hydrogen bonds, and covalent bonds (Moore,W. J., In: Physical Chemistry, 4th Ed., Prentice Hall, (1-972), pp.865-898).

[0207] Thus, in many ways protein crystallization can be viewed as ahigher level variation of protein folding where whole molecules arepacked to maximize cohesive energies instead of individual amino acidresidues. Moreover, for both protein crystallization and proteinfolding, the composition of the solvent can make very importantcontributions to the extent of partitioning between the soluble(unfolded) and crystalline (native) forms. The cohesive interactionspresent in protein macromolecules and the role played by solvent inmodulating these interactions for both protein folding and proteincrystallization are complex and not fully understood at the presenttime. In this regard, biochemical conditions that promote proteinstabililty and protein folding also promote protein crystallization.

[0208] For example, biochemical conditions that were found to increasethe stability of D(II) FGF receptor 1 (FIGS. 19-24) correlate with theconditions that facilitated the crystallization of x-ray diffractionquality protein crystals. Conditions that were employed to obtaincrystals of D(II) FGFR1 protein (Lewankowski, Myslik, Bone, R. Springer,B. A. and Pantoliano, M. W., unpublished results (1997)) are shown inTable 1. Protein crystals were obtained in the pH range 7.4 to 9.2 inthe presence of the Hofmeister salt Li₂SO₄ (65 to 72%). Thesecrystallization conditions correlated with the pH optimum of about 8.0in FIG. 23. Other salts of the Hofmeister series such as Na₂SO₄,(NH₄)₂SO₄ and Mg₂SO₄ were also found useful as additives for loweringthe amount of Li₂SO₄ required as the precipitant. Clearly, theseconditions for successful D(II) FGFR1 crystallization correlate closelywith the stabilizing conditions that were identified using themicroplate thermal shift assay.

[0209] Conditions that were identified as facilitating human α-thrombinstabilization also facilitate human α-thrombin protein crystallization.FIGS. 17A-D and 18 show the results of microplate thermal shift assaysof conditions that facilitate human α-thrombin stability. Table 2contains a summary of the conditions identified by three differentinvestigators that facilitate crystallization of x-ray diffractionquality human α-thrombin crystals (Bode, W., et al., Protein Sci.1:426-471 (1992); Vijayalakshmi, J. et al., Protein Sci. 3:2254-22271(1994); and Zdanov, A. et al., Proteins: Struct. Funct. Genet.17:252-265 (1993)).

[0210] The conditions summarized in Table 2 correlate closely with theconditions identified in the microplate thermal shift assay asfacilitating human α-thrombin stability. Crystals formed near a pHoptimum of about 7.0. Furthermore, there is a clear preference for thepresence of 0.1 to 0.5 M NaCl (50% of the conditions) or 0.1 to 0.2 MNaHPO₄. This is consistent with the recently discovered Na⁺ binding site(Dang et al., Nature Biotechnology 15:146-149 (1997)) and microplatethermal shift assay results in FIGS. 17A-D and 18. All of the humanα-thrombin samples described in Table 2 that have yielded good crystalsare complexed with a ligand, thereby further stabilizing the nativestructure of this protein beyond that acquired from the biochemicalconditions. TABLE 1 D(II) FGFR1 Crystallization Conditions BufferPrecipitant Additive Protein Concentration 50 mM Hepes pH 7.4 72% Li2SO410 mg/ml (10 mM Hepes pH 7.5) 50 mM Hepes pH 7.4 72% Li2SO4 3.4 mM ZnSO410 mg/ml (10 mM Hepes pH 7.5) 50 mM Hepes pH 7.4 68% Li2SO4 1% PEG 800010 mg/ml (10 mM Hepes pH 7.5) 50 mM Hepes pH 7.4 66% Li2SO4 3.4 mMNa2SO4 10 mg/ml (10 mM Hepes pH 7.5) 50 mM Hepes pH 7.4 66% Li2SO4 5.3mM (NH4) 2SO4 10 mg/ml (10 mM Hepes pH 7.5) 50 mM Hepes pH 7.4 66%Li2SO4 2.1 mM MgSO4 10 mg/ml (10 mM Hepes pH 7.5) 10 mM Tris Hcl, 65%Li2SO4 10 mg/ml (10 mM Hepes pH 7.5) pH 8.0 20 mM glycine, 68% Li2SO4 10mg/ml (10 mM Hepes pH 7.5) pH 5.2

[0211] Protein crystallization is a slow and tedious process that hashistorically been the rate determining step for the x-ray diffractiondetermination of protein and nucleic acid structures. The method andapparatus of the present invention facilitate the rapid, high-throughputelucidation of conditions that promote the stability of a given proteinand thus the formation of X-ray quality protein crystals.

[0212] When a protein is more stable, it has fewer thermodynamic motionsthat inhibit packing into a crystal lattice. With fewer motions, theprotein fits better into a crystal lattice. Using conventionalcrystallization methods, crystallization experiments are set up at roomtemperature for weeks at a time. Over time, protein unfolding occurs.Using the methods of the present invention, conditions that stabilize aprotein are examined over a temperature range. Conditions that shift thethermal unfolding curve to higher temperature will lower extent ofunfolding that occurs while the crystallization process occurs. TABLE 2Human α- Crystallization Conditions thrombin Protein Complex Buffer SaltPrecipitant Additive Conc. Comment Vijayalakshmi et al. MDL-28050 75 mMNaHPO4 pH 7.3 0.375 M NaCl 1 mM NaN3 3 mg/ml protein (2.2 Å) 100 mMNaHPO4 pH 7.3 24% PEG 4000 1 mM NaN3 well Hirugen/Hirulog 1 50 mM NaHPO4pH 7.3 0.375 M NaCl .5 mM NaN3 3-3.7 mg/ml protein (2.3 Å) 0.1 M NaHPO4pH 7.3 28% PEG 8000 1 mM NaN3 well FPAM + Hirugen 0.1 M NaHPO4 pH 7.3 5mg/ml protein (2.5 Å) 0.1 M NaHPO4 pH 7.3 28% PEG 8000 well Hirulog 3 75mM NaHPO4 pH 7.3 0.38 M NaCl 1 mM NaN3 5 mg/ml protein (2.3 Å) 0.1 MNaHPO4 pH 7.3 24% PEG 8000 1 mM NaN3 well Bode et al. NAPAP 0.1 M KHPO4pH 8.0 10 mg/ml protein (2.3 Å) 0.1 M KHPO4 pH 8.0 1.9 M NH4SO4 PPACK 2mM MOPS pH7  0.1 M NaCl 0.5% NaN3 10 mg/ml protein (1.9 Å) 0.2 M P04 pH6-7  0.5 M NaCl 20% PEG 6000 well Zdanov et al. Hirutonin-2 50 mM NaHPO4pH 5.5  0.1 M NaCl 10 mg/ml protein (2.1 Å) 0.1 M Na Citrate pH 5.5  0.1M NaCl 24% PEG 4000 well

Overview of Assay Apparatus

[0213] The assay apparatus of the present invention is directed to anautomated temperature adjusting and spectral emission receiving systemthat simultaneously adjusts the temperature of a multiplicity of samplesover a defined temperature range and receives spectral emission from thesamples. The assay apparatus of the present invention is particularlyuseful for performing microplate thermal shift assays of proteinstability. The assay apparatus of the present invention can be used topractice all of the methods of the present invention.

[0214] The assay apparatus of the present invention replaces separateheating devices and spectral emission receiving devices. In contrast toother devices, the assay apparatus of the present invention can beconfigured to simultaneously adjust the temperature of a multiplicity ofsamples and receive spectral emissions from the samples duringadjustment of temperature in accordance with a predetermined temperatureprofile.

[0215] After heat denaturation, reversibly folding proteins partially orfully refold after heat denaturation. Refolding precludes meaningfulmeasurements in a thermal shift assay. Using the assay apparatus of thepresent invention, however, one can assay reversibly folding proteins ina thermal shift assay. That is because in the assay apparatus of thepresent invention, spectral measurements are taken while the protein isbeing heated. And protein refolding does not occur.

[0216] In such a configuration, the assay apparatus of the presentinvention includes a sensor which is positioned over a movable heatconducting block upon which an array of samples is placed. A relativemovement means, such as a servo driven armature, is used to move thesensor so that the sensor is sequentially positioned over each sample inthe array of samples. The sensor receives spectral emissions from thesamples.

[0217] The assay apparatus of the present invention can be configured sothat it contains a single heat conducting block. Alternatively, theassay apparatus can be configured so that it contains a plurality ofheat conducting blocks upon a movable platform. The platform may be atranslatable platform that can be translated, for example, by a servodriven linear slide device. An exemplary linear slide device is model SAA5M400 (IAI America, Torrance, Calif.). In this embodiment, the sensorreceives spectral emissions from each of the samples on a given heatconducting block. The platform is then translated to place another heatconducting block and its accompanying samples under the sensor so thatit receives spectral emissions from each of the samples on that heatingblock. The platform is translated until spectral emissions are receivedfrom the samples on all heat conducting blocks.

[0218] Alternatively, the platform may by a rotatable platform that maybe rotated, for example, by a servo driven axle. In the latterembodiment, the sensor receives spectral emissions from each of thesamples on a given heat conducting block. The platform is then rotatedto place another heat conducting block and its accompanying samplesunder the sensor so that it receives spectral emissions from each of thesamples on that heating block. The platform is rotated until spectralemissions are received from the samples on all heat conducting blocks.

System Description

[0219]FIG. 29 shows a schematic diagram of one embodiment of an assayapparatus 2900 of the present invention. Assay apparatus 2900 includes aheat conducting block 2912 that includes a plurality of wells 2920 for aplurality of samples 2910. Heat conducting block 2912 is composed of amaterial that has a relatively high coefficient-of thermal conductivity,such as aluminum, stainless steel, brass, teflon, and ceramic.

[0220] Thus, heat conducting block 2912 can be heated and cooled to auniform temperature but will not be thermally conductive enough torequire excess heating or cooling to maintain a temperature.

[0221] Assay apparatus 2900 also includes a light source 2906 foremitting an excitatory wavelength of light, shown generally at 2916, forthe samples. Light source 2906 excites samples 2910 with excitatorylight 2916. Any suitable light source can be used. For example atungsten-halogen lamp can be used. Alternatively, a Xenon-arc lamp, suchas the Biolumin 960 (Molecular Dynamics) can used.

[0222] Alternatively, a high pressure mercury (Hg) Lamp can be used.High pressure mercury lamps emit light of higher intensity than Xenon(Xe) lamps. The intensity of light from a high pressure mercury lamp isconcentrated in specific lines, and are only useful if the Hg lines areat suitable wavelengths for excitation of particular fluorophores.

[0223] Some fluorescent plate readers employ lasers for excitation inthe visible region of the electromagnetic spectrum. For example, theFluorImager™ (Molecular Dynamics, Palo Alto, Calif.) is such a device.This technology is useful when using fluorescent dyes that absorb energyat around 480 nm and emit energy at around 590 nm. Such a dye could thenbe excited with the 488 nm illumination of standard argon, argon/kryptonlasers. For example,1,1-dicyano-2-[6-(di-methylamino)naphthalen-2-yl]propene (DDNP) is sucha dye. The advantage in using a laser is that a laser is characterizedby very high intensity light, which results in an improved signal tonoise ratio.

[0224] Excitatory light 2916 causes a spectral emission 2918 fromsamples 2910. Spectral emission 2918 can be electromagnetic radiation ofany wavelength in the electromagnetic spectrum. Preferably, spectralemission 2918 is fluorescent, ultraviolet, or visible light. Mostpreferably, spectral emission 2918 is fluorescence emission. Spectralemission 2918 is received by a photomultiplier tube 2904.Photomultiplier tube 2904 is communicatively and operatively coupled toa computer 2914 by an electrical connection 2902. Computer 2914functions as a data analysis means for analyzing spectral emission as afunction of temperature.

[0225] As discussed above, the spectral receiving means or sensor of theassay apparatus of the present invention can comprise a photomultipliertube. Alternatively, the spectral receiving means or sensor can includea charge coupled device or a charge coupled device camera. In stillanother alternative, the spectral receiving means or sensor can includea diode array.

[0226] An alternate embodiment of the assay apparatus of the presentinvention is shown in FIG. 30. In the embodiment shown in FIG. 30, acharge coupled device (CCD) camera 3000 is used to detect spectralemission 2918 from samples 2910. CCD camera 3000 can be any CCD camerasuitable for imaging fluorescent emissions. For example, suitable CCDcameras are available from Alpha-Innotech (San Leandro, Calif.),Stratagene (La Jolla, Calif.), and BioRad (Richmond, Calif.). Formeasuring fluorescent emission in the microplate thermal shift assay,one alternative to a fluorescent plate reader is a charge coupled device(CCD). For example, high resolution CCD cameras can detect very smallamounts of electromagnetic energy, whether it originates from distancestars, is diffracted by crystals, or is emitted by fluorophores. A CCDis made of semi-conducting silicon. When photons of light fall on it,free electrons are released. As an electronic imaging device, a CCDcamera is particularly suitable for fluorescence emission imagingbecause it can detect very faint objects, affords sensitive detectionover a broad spectrum range, affords low levels of electromagneticnoise, and detects signals over a wide dynamic range—that is, a chargecoupled device can simultaneously detect bright objects and faintobjects. Further, the output is linear so that the amount of electronscollected is directly proportional to the number of photons received.This means that the image brightness is a measure of the real brightnessof the object, a property not afforded by, for example, photographicemulsions.

[0227] When a fluorescence imaging camera or a CCD camera is used,excitatory light 2916 can be a suitable lamp that is positioned over theplurality of samples 2910. Alternatively, excitatory light 2916 can be asuitable lamp that is positioned under the plurality of samples 2910. Inanother alternative embodiment, excitatory light 2916 can be deliveredto each sample 2910 by a plurality of fiber optic cables. Each fiberoptic cable is disposed through one of a plurality of tunnels inconducting block 2912. Thus, each of samples 2910 receives excitatorylight 2916 through a fiber optic cable.

[0228] As shown in FIG. 30, source 2906 excites samples 2910 withexcitatory light 2916. Excitatory light 2916 causes spectral emission2918 from samples 2910. Spectral emission 2918 is filtered through anemission filter 3002. Emission filter 3002 filters out wavelengths ofspectral emission 2918 that are not to be monitored or received by CCDcamera 3000. CCD camera 3000 receives the filtered spectral emission2918 from all of samples 2910 simultaneously. For simplicity and ease ofunderstanding, only spectral emissions form one row of samples 2910 areshown in FIG. 30. CCD camera 3000 is communicatively and operativelycoupled to computer 2914 by electrical connection 2902.

[0229] With reference now to FIG. 31, one embodiment of assay apparatus2900 is shown in more detail. As shown in FIG. 31, many apparatuscomponents are attached to a base 3100. A heat conducting block relativemovement means 3128 is used to move heat conducting block 2912 indirections 3150 and 3152. Heat conducting block relative movement means3128 is communicatively and operatively connected to a servo controller3144. Activation of heat conducting block relative movement means 3128by servo controller 3144 moves heat conducting block 2912 in directions3150 and 3152. Servo controller 3144 is controlled by a computercontroller 3142. Alternatively, computer 2914 could be used to controlservo controller 3144.

[0230] A sensor is removably attached to a sensor armature 3120. Anexemplary sensor is a fiber optic probe 3122. Fiber optic probe 3122includes a fiber optic cable capable of transmitting receivingexcitatory light 2916 to samples 2910, and a fiber optic cable capableof receiving spectral emission 2918 from samples 2910. Electromagneticradiation is transmitted from excitatory light source 2906 to fiberoptic probe 3122 by excitatory light input fiber optic cable 3108. Inone embodiment of the present invention, a spectral receiving meanscomprising photomultiplier tube 2904 is used to detect spectral emissionfrom samples 2910. In this embodiment, electromagnetic radiation istransmitted from fiber optic probe 3122 to photomultiplier tube 2904 byfiber optic cable 3110. In an alternative embodiment of the presentinvention, CCD camera 3002 is used to detect spectral emission fromsamples 2910. In this embodiment, fiber optic cable 3110 is notrequired.

[0231] A temperature sensor 3124 is removably attached to sensorarmature 3120. Temperature sensor 3124 is communicatively and operablylinked to a temperature controller 3162. Temperature sensor 3124monitors the temperature of heat conducting block 2912 and feedstemperature information back to temperature controller 3162. Temperaturecontroller 3162 is connected to heat conducting block 2912 by athermoelectric connection 3164. Under the action of temperaturecontroller 3162, the temperature of heat conducting block 2912 can beincreased, decreased, or held constant. Particularly, the temperature ofheat conducting block 2912 can be changed by temperature controller 3162in accordance with a pre-determined temperature profile. Preferably,temperature computer controller 3162 is implemented using a computersystem such as that described below with respect to FIG. 37.

[0232] As used herein, the term “temperature profile” refers to a changein temperature over time. The term “temperature profile” encompassescontinuous upward or downward changes in temperature, both linear andnon-linear changes. The term also encompasses any stepwise temperaturechange protocols, including protocols characterized by incrementalincreases or decreases in temperature during which temperature increasesor decreases are interrupted by periods during which temperature ismaintained constant. In the apparatus of the present invention, thetemperature profile can be pre-determined by programming temperaturecomputer controller 3162. For example, temperature profiles can bestored in a memory device of temperature controller 3162, or input totemperature controller 3162 by an operator.

[0233] A sensor armature relative movement means 3130 is used to movesensor armature 3120 in directions 3154 and 3156. A sensor armatureservo controller 3118 is fixedly connected to excitatory light filterhousing 3160. Activation of sensor armature servo controller 3118 movesfiber optic probe 3122 in directions 3154 and 3156. It would be readilyapparent to one of ordinary skill in the relevant art how to configureservo controllers to move heat conducting block 2912 and sensor armature3120. It should be understood that the present invention is not limitedto the use of servo controllers for movement of heat conducting block2912 and sensor armature 3120, and other suitable means known to one ofskill in the art can also be used, such as a motor.

[0234] Servo controllers 3118 and 3144 are both communicatively andoperatively connected to computer controller 3142. Computer controller3142 controls the movement of sensor armature 3120 in directions 3154and 3156. In addition, computer controller 3142 controls the movement ofheat conducting block relative movement means 3128 in directions 3150and 3152.

[0235] In the assay apparatus of the present invention, excitatory lightsource 2906 is used to excite samples 2910. Excitatory light source 2906is communicatively and operably connected to excitatory light filter3104, which is contained within excitatory light filter housing 3160.Excitatory light filter 3104 filters out all wavelengths of light fromexcitatory light source 2906 except for the wavelength(s) of light thatare desired to be delivered by fiber optic probe 3122 to samples 2910.An excitatory light filter servo controller 3106 controls the apertureof excitatory light filter 3104. Excitatory light source 2906 andexcitatory light filter servo controller 3106 are communicatively andoperatively connected to excitatory light computer controller 3102.Computer controller 3102 controls the wavelength of excitatory lighttransmitted to samples 2910 by controlling excitatory light filter servocontroller 3106. Excitatory light 2916 is transmitted through excitatorylight input fiber optic cable 3108 to fiber optic probe 3122 fortransmission to samples 2912.

[0236] Spectral emission :2918 from samples 2910 is received by fiberoptic probe 3122 and is transmitted to a spectral emission filter 3114by output fiber optic cable 3110. Spectral emission filter 3114 iscontained within a spectral emission filter housing 3166. Spectralemission filter housing 3166 is disposed on photomultiplier tube housing3168. Photomultiplier tube housing 3168 contains photomultiplier tube2904. A spectral emission servo controller 3112 controls the aperture ofspectral emission filter 3114, thereby controlling the wavelength ofspectral emission 2918 that is transmitted to photomultiplier tube 2904.Spectral emission servo controller 3112 is controlled by a computercontroller 3170.

[0237] Spectral emission 2918 from samples 2910 is transmitted fromphotomultiplier tube 2904. Electrical output 3140 connectsphotomultiplier tube 2904 to electric connection 2902. Electricconnection 2902 connects electrical output 3140 to computer 2914. Drivenby suitable software, computer 2914 processes the spectral emissionsignal from samples 2910. Exemplary software is a graphical interfacethat automatically analyzes fluorescence data obtained from samples2910. Such software is well known to those of ordinary skill in the art.For example, the CytoFluor™II fluorescence multi-well plate reader(PerSeptive Biosystems, Framingham, Mass.) utilizes the Cytocalc™ DataAnalysis System (PerSeptive Biosystems, Framingham, Mass.). Othersuitable software includes, MicroSoft Excel or any comparable software.

[0238] FIGS. 32A-C illustrate one embodiment of a thermal electric stageor heat conducting block for the assay apparatus of the presentinvention. FIG. 32A shows a side view of heat conducting block 2912 anda heat conducting wire 3206. FIG. 32B shows a top view of heatconducting block 2912 and heat conducting wire 3206. Heat conductingwire 3206 is a temperature adjusting element that adjusts thetemperature of heat conducting block 2912. By means readily known to oneof skill in the art, temperature controller 3162 causes heat conductingwire 3206 to increase or decrease in temperature, thereby changing thetemperature of heat conducting block 2912. For example, an exemplarytemperature controller is a resistance device that converts electricenergy into heat energy. Alternatively, the heating element can be acirculating water system, such as that disclosed in U.S. Pat. No.5,255,976, the content of which is incorporated herein by reference. Inanother alternative, the temperature adjusting element can be a heatconducting surface upon which heat conducting block 2912 is disposed.Particularly, the temperature of heat conducting wire 3206 can bechanged by temperature controller 3162 in accordance with apre-determined temperature profile. Temperature controller 3162 ispreferably implemented using a computer system such as that describedbelow with respect to FIG. 37. Alternatively, computer 2914 could beused to implement temperature controller 3162. An exemplary set ofspecifications for temperature controller 3162 and heat conducting block2912 is as follows: resolution 0.1° C. accuracy +0.5° C.  stability 0.1°C. repeatability 0.1° C.

[0239] Temperature controller 3162 changes temperature in accordancewith a temperature profile as discussed below with respect to FIGS. 36Aand 36B.

[0240] The temperature of heat conducting block 2912 can be controlledsuch that a uniform temperature is maintained across the heat conductingblock. Alternatively, the temperature of heat conducting block 2921 canbe controlled such that a temperature gradient is established from oneend of the heat conducting block to the other. Such a technique isdisclosed in U.S. Pat. Nos. 5,255,976 and 5,525,300, the entirety ofboth of which is incorporated by reference.

[0241] Heat conducting block 2912 is preferably configured withplurality of wells 2920 for samples 2910 to be assayed. In oneembodiment, each of wells 2920 is configured to receive a containercontaining one of plurality of samples 2910. Alternatively, heatconducting block 2912 is configured to receive a container containingplurality of samples 2910. An exemplary container for containingplurality of samples 2910 is a microtiter plate.

[0242] In yet a further alternate embodiment, heat conducting block 2912is configured to receive a heat conducting adaptor that is configured toreceive a container containing one or more of samples 2910. The heatconducting adaptor is disposed on heat conducting block 2912, and thecontainer containing samples 2910 fits into the heat conducting adaptor.FIGS. 32C-E show three exemplary configurations of a heat conductingadaptor. An adaptor 3200 is configured with round-bottomed wells. Anadaptor 3202 is configured with square-bottom wells. An adaptor 3204 isconfigured with V-shaped wells. For example, adaptor 3200 can receive aplurality of round-bottom containers, each containing one sample.Similarly, adaptor 3202 can receive a plurality of square-bottomcontainers, and adaptor 3204 can contain a plurality of V-shaped bottomcontainers. Adaptors 3200, 3202, and 3204 can also receive a carrier fora multiplicity of round-bottom containers. An exemplary carrier is amicrotitre plate having a plurality of wells, each well containing asample. When heat conducting block 2912 is heated, heat conductingadaptors 3200, 3202, or 3204 are also heated. Thus, the samplescontained in the containers that fit within adaptors 3200, 3202, or 3204are also heated. Adaptors 3200, 3202, and 3204 can accept standardmicroplate geometries.

[0243] Another embodiment of the assay apparatus of the presentinvention is shown in FIG. 33. In this embodiment a plurality of heatconducting blocks 2912 is mounted on a rotatable platform or carousel3306. Alternativeley, the platform can be a translatable platform.Platform or carousel 3306 can be composed of a heat conducting material,such as the material that heat conducting block 2912 is composed of.Although six heat conducting blocks are shown in FIG. 33, this number isexemplary and it is to be understood that any number of heat conductingblocks can be used. As shown in FIG. 33, an axle 3308 is rotatablyconnected to base 3100. Rotatable platform 3306 is axially mounted torotate about axle 3308. Rotation of axle 3308 is controlled by a servocontroller 3312. Servo controller 3312 is controlled by a computercontroller 3314 in a manner well known to one of skill in the relevantarts. Computer controller 3314 causes servo controller 3312 to rotateaxle 3308 thereby rotating rotatable platform 3306. In this manner, heatconducting blocks 2912 are sequentially placed under fiber optic probe3122.

[0244] Each of the plurality of heat conducting blocks 2912 can becontrolled independently by temperature controller 3162. Thus, thetemperature of a first heat conducting block 2912 can be higher or lowerthan the temperature of a second heat conducting block 2912. Similarly,the temperature of a third heat conducting block 2912 can be higher orlower than the temperature of either first or second heat conductingblock 2912.

[0245] In a manner similar to that described above for FIG. 31, relativemovement means 3130 is also used to move sensor armature 3120 indirections 3150 and 3152 so that fiber optic probe 3122 can be moved todetect spectral emission from samples 2910. A second sensor armaturerelative movement means 3316 is used to move sensor armature 3120 indirections 3154 and 3156.

[0246] The temperature of heat conducting blocks 2912 is controlled bytemperature controller 3162. Temperature controller 3162 is connected torotatable platform 3306 by connection 3164 to heat conducting blocks2912. Under the action of temperature controller 3162, the temperatureof heat conducting blocks 2912 can be increased and decreased.Alternatively, temperature controller 3162 can be configured to adjustthe temperature of rotatable platform 3306. In such a configuration,when rotatable platform 3306 is heated, heat conducting blocks 2912 arealso heated. Alternatively, the temperature of each of heat conductingblocks 2912 can be controlled by a circulating water system such as thatnoted above.

[0247] In a manner similar to that illustrated in FIG. 31, excitatorylight source 2906 is used to excite samples 2910. Excitatory lightsource 2906 is communicatively and operably connected to excitatorylight filter 3104, which is contained within excitatory light filterhousing 3160. Excitatory light filter 3104 filters out all wavelengthsof light from excitatory light source 2906 except for the wavelength(s)of light that are desired to be delivered by fiber optic probe 3122 tosamples 2910. An excitatory light filter servo controller 3106 controlsthe aperture of excitatory light filter 3104. Excitatory light source2906 and excitatory light filter servo controller 3106 arecommunicatively and operatively connected to excitatory light computercontroller 3102. Computer controller 3102 controls the wavelength ofexcitatory light transmitted to samples 2910 by controlling excitatorylight filter servo controller 3106. Excitatory light 2916 is transmittedthrough excitatory light input fiber optic cable 3108 to fiber opticprobe 3122 for transmission to samples 2912.

[0248] Spectral emission 2918 from samples 2910 is received by fiberoptic probe 3122 and is transmitted to spectral emission filter 3114 byfiber optic cable 3110. Spectral emission servo controller 3112 controlsspectral emission filter 3114 aperture and thus controls the wavelengthof spectral emission that is transmitted to photomultiplier tube 2904.In a manner similar to that explained for FIG. 31, spectral emissionservo controller 3112 is controlled by computer controller 3170.

[0249] The assay apparatus of the present invention can detect spectralemission from samples 2910 one sample at a time or simultaneously from asubset of samples 2910. As used herein, the term “subset of samples”refers to at least two of samples 2910. To detect spectral emissionsimultaneously from a subset of samples in an embodiment of the assayapparatus of the present invention comprising photomultiplier tube 2904,a plurality of excitatory light filters 3104, excitatory light inputfiber optic cables 3108, emission light output fiber optic cables 3110,and emission light filters 3114 must be used.

[0250] The spectral emission signal is transmitted from photomultipliertube 2904 to computer 2914. Photomultiplier tube 2904 is communicativelyand operatively coupled to computer 2914 by electrical connection 2902.Connection 2902 is connected to photomultiplier tube 2904 throughelectrical output 3140. Computer 2914 functions as a data analysis meansfor analyzing spectral emission as a function of temperature.

[0251]FIG. 34 illustrates a top view of the assay apparatus shown inFIG. 33 with a housing 3400 that covers the apparatus. A door 3402 opensto reveal samples 2910. Door 3402 can be a hinge door that swings open.Alternatively, door 3402 can be a sliding door that slides open. A sideview of the assay apparatus shown in FIGS. 33 and 34 is illustrated inFIG. 35. Cover 3400 is disposed on top of base 3100. Cover 3400 can bemade of any suitable material. For example, cover 3400 can be made ofplexiglass, fiberglass, or metal.

[0252]FIGS. 36A and 36B illustrate a temperature profile and how thetemperature profile is implemented using the assay apparatus of thepresent invention. FIG. 36A illustrates a temperature profile 3600 thatshows the temperature of heat conducting blocks 2912 as a function oftime. Heat conducting blocks 2912 and samples 2910 are heated in acontinuous fashion in accordance with temperature profile 3600.Alternatively, rotatable platform 3306 can be heated along with heatconducting blocks 2912: Preferably, temperature profile 3600 is linear,with temperatures ranging from about 25° C. to about 110° C.

[0253] Alternatively, temperature profile 3600 can be characterized byincremental, stair step increases in temperature, in which heatconducting blocks 2912 and samples 2910 are heated to a predeterminedtemperature, maintained at that temperature for a predetermined periodof time, and than heated to a higher predetermined temperature. Forexample, temperature can be increased from 0.5° C. to 20° C. per minute.Although the temperature range from about 25° C. to about 110° C. isdisclosed, it is to be understood that the temperature range with whicha given target molecule, for example, a protein, is to be heated togenerate a thermal denaturation curve can readily be determined by oneof ordinary skill in the art. The length of time over which temperatureprofile 3600 is accomplished will vary, depending on how many samplesare to be assayed and on how rapidly the sensor that receives spectralemission 2918 can detect spectral emission 2918 from samples 2910. Forexample, an experiment in which each of six heat conducting blocks 2912holds a total of 96 samples 2910 (for a total of 576 samples), and inwhich samples are scanned using a fluorescent reader device having asingle fiber optic probe, and in which the temperature profile is from38° C. and 76° C., would take approximately 38 minutes to perform usingthe apparatus shown in FIG. 33.

[0254] While heating in accordance with temperature profile 3600,spectral emission 2918 from each sample 2910 in a first heat conductingblock 2912 is received through fiber optic probe 3122. As illustrated inFIG. 36B, after emissions from all of samples 2910 in first heatconducting block 2912 have been received, platform 3306 is rotated tomove the next heat conducting block 2912 under fiber optic probe 3122and spectral emission 2918 from samples 2910 is received by fiber opticprobe 3122. This process is continued until reception of spectralemissions from all samples in all heat conducting blocks 2912 iscomplete. Spectral emission from samples 2910 on each heat conductingblock 2912 can be received one at a time, simultaneously from a subsetof samples, simultaneously from one row of samples at a time, or all ofthe samples at one time.

Computer Program Implementation of the Preferred Embodiments

[0255] The present invention may be implemented using hardware,software, or a combination thereof, and may be implemented in a computersystem or other processing system. A flowchart 3800 for implementationof one embodiment of the present invention is shown in FIG. 38.Flowchart 3800 begins with a start step 3802. In a step 3804,temperature profile 3600 is initiated. For example, temperaturecontroller 3162 causes the temperature of heat conducting block 2912 toincrease. In a step 3806, a sensor such as fiber optic probe 3122 or CCDcamera 3000 is moved over a sample 2910, row of samples 2910, or all ofsamples 2910. In a step 3808, excitatory light is transmitted tosample(s) 2910 using excitatory light source 2906. In a step 3810,spectral emission is received by the sensor from sample(s) 2910. In adecision step 3812, it is determined whether spectral emission 2918 hasbeen received from all of the samples, rows of samples, in one heatconducting block 2912. If spectral emission 2918 has not been receivedfrom all of the samples or rows of samples, the sensor is moved over thenext sample or row of samples in a step 3814. Processing then continuesat step 3808 to transmit excitatory light 2916. Processing thencontinues to a step 3810 to receive spectral emission 2918 fromsample(s) 2910.

[0256] If spectral emission 2918 has been received from all of samplesor rows of samples, processing continues to a decision step 3816. Indecision step 3816, it is determined whether spectral emission 2918 hasbeen received from samples in all heat conducting blocks. If not,rotatable platform 3306 is rotated in a step 3818 to place the next heatconducting block 2912 and samples 2910 contained therein under thesensor. Steps 3806 through 3818 are followed until spectral emission2918 has been received from all of the samples in all of heat conductingblocks 2912. Processing then continues to a step 3820, in whichtemperature profile 3600 is completed and processing ends at a step3822.

[0257] A flowchart 3900 for implementation of an alternate embodiment ofthe present invention is shown in FIG. 39. In this embodiment, a sensorfor simultaneously receiving spectral emission 2918 from all of samples2910 on heat conducting block 2912, such as CCD camera 3000, ispositioned over heat conducting block 2912. Flowchart 3900 begins with astart step 3902. In a step 3904, temperature profile 3600 is initiated.For example, temperature controller 3162 causes the temperature of heatconducting block 2912 to increase. In a step 3906, excitatory light istransmitted to sample(s) 2910 using excitatory light source 2906. In astep 3908, spectral emission is received by CCD camera 3000 fromsample(s) 2910. In a decision step 3910, it is determined whetherspectral emission 2918 has been received from all of heat conductingblocks 2912. If not, rotatable platform 3306 is rotated in a step 3912to place the next heat conducting block 2912 and samples 2910 containedtherein under CCD camera 3000. Steps 3906 through 3912 are followeduntil spectral emission 2918 has been received from samples 2910 in allof heat conducting blocks 2912. Processing then continues to a step3914. In step 3914, temperature profile 3600 is completed and processingends at a step 3916.

[0258] As stated above, the present invention may be implemented usinghardware, software, or a combination thereof, and may be implemented ina computer system or other processing system. An exemplary computersystem 3702 is shown in FIG. 37. Computer controllers 3102, 3142, 3162,3170, or 3314, can be implemented using one or more computer systemssuch as computer system 3702.

[0259] After reading this description, it will become apparent to aperson skilled in the relevant art how to implement the invention usingother computer systems and/or computer architectures. Computer system3702 includes one or more processors, such as processor 3704. Processor3704 is connected to a communication bus 3706.

[0260] Computer system 3702 also includes a main memory 3708, preferablyrandom access memory (RAM), and can also include a secondary memory3710. The secondary memory 3710 can include, for example, a hard diskdrive 3712 and/or a removable storage drive 3714, representing a floppydisk drive, a magnetic tape drive, an optical disk drive, etc. Theremovable storage drive 3714 reads from and/or writes to a removablestorage unit 3716 in a well known manner. Removable storage unit 3716represents a floppy disk, magnetic tape, optical disk, etc. which isread by and written to by removable storage drive 3714. As will beappreciated, the removable storage unit 3716 includes a computer usablestorage medium having stored therein computer software and/or data.

[0261] In alternative embodiments, secondary memory 3710 may includeother similar means for allowing computer programs or other instructionsto be loaded into computer system 3702. Such means can include, forexample, a removable storage unit 3718 and an interface 3720. Examplesof such can include a program cartridge and cartridge interface (such asthat found in video game devices), a removable memory chip (such as anEPROM, or PROM) and associated socket, and other removable storage units3718 and interfaces 3720 which allow software and data to be transferredfrom the removable storage unit 3718 to computer system 3702.

[0262] Computer system 3702 can also include a communications interface3722. Communications interface 3722 allows software and data to betransferred between computer system 3702 and external devices. Examplesof communications interface 3722 can include a modem, a networkinterface (such as an Ethernet card), a communications port, a PCMCIAslot and card, etc. Software and data transferred via communicationsinterface 3722 are in the form of signals 3724 which can be electronic,electromagnetic, optical or other signals capable of being received bycommunications interface 3722. These signals 3724 are provided tocommunications interface via a channel-3726. This channel 3726 carriessignals 3724 and can be implemented using wire or cable, fiber optics, aphone line, a cellular phone link, an RF link and other communicationschannels. In the assay apparatus of the present invention, one exampleof channel 3726 is electrical connection 2902 that carries signal 3724of spectral emission 2918 to computer 2914.

[0263] In this document, the terms “computer program medium” and“Computer usable medium” are used to generally refer to media such asremovable storage device, 3716 and 3718, a hard disk installed in harddisk drive 3712, and signals 3724. These computer program products aremeans for providing software to computer system 3702.

[0264] Computer programs (also called computer control logic) are storedin main memory 3708 and/or secondary memory 3710. Computer programs canalso be received via communications interface 3722. Such computerprograms, when executed, enable the computer system 3702 to perform thefeatures of the present invention as discussed herein. In particular,the computer programs, when executed, enable the processor 3704 toperform the features of the present invention. Accordingly, suchcomputer programs represent controllers of the computer system 3702.

[0265] In an embodiment where the invention is implemented usingsoftware, the software may be stored in a computer program product andloaded into computer system 3702 using removable storage drive 3714,hard drive 3712 or communications interface 3722. The control logic(software), when executed by the processor 3704, causes the processor3704 to perform the functions of the invention as described herein.

[0266] In another embodiment, the invention is implemented primarily inhardware using, for example, hardware components such as applicationspecific integrated circuits (ASICs). Implementation of the hardwarestate machine so as to perform the functions described herein will beapparent to persons skilled in the relevant art(s).

[0267] In yet another embodiment, the invention is implemented using acombination of both hardware and software.

[0268] The assay apparatus of the present invention is particularlysuited for carrying out the methods of the present invention. To conducta microplate thermal shift assay using the method and apparatus of thepresent invention, samples are placed in a heat conducting block, heatedaccording to a predetermined temperature profile, stimulated with anexcitatory wavelength of light, and the spectral emission from thesamples is detected while the samples are being heated in accordancewith the pre-determined temperature profile.

[0269] It is to be understood that the assay apparatus of the presentinvention is not limited to use with the methods of the presentinvention or limited to conducting assays on biological polymers,proteins, or nucleic acids. For example, the assay apparatus of thepresent invention can be used to incubate samples to a predeterminedtemperature. Alternatively, the assay apparatus of the present inventioncan be used to perform polymerase chain reaction, thermal cycling stepsfor any purpose, assaying thermal stability of a compound, such as adrug, to determine conditions that stabilize a compound, or to determineconditions that facilitate crystallization of a compound.

[0270] Having now generally described the invention, the same willbecome more readily understood by reference to the following specificexamples which are included herein for purposes of illustration only andare not intended to be limiting unless otherwise specified.

EXAMPLE 1 Ranking Ligands that Bind to the Active Site of Humanα-Thrombin

[0271] Using the computer controlled process DirectedDiversity® (seeU.S. Pat. No. 5,463,564), scientists at 3-Dimensional Pharmaceuticals,Inc. have generated a combinatorial library of compounds directed at theactive site of human α-thrombin. Approximately 400 compounds weresynthesized and assayed by a conventional spectrophotometric kineticassay in which succinyl-Ala-Ala-Pro-Arg-p-nitroanilide (Bachem, King ofPrussia, Pa.) served as substrate. Five of these compounds, which arecharacterized by K_(i)'s that span almost four orders of magnitude inbinding affinity, were used to test the range and limits of detection ofthe thermal shift assay. These five proprietary compounds are listed inTable 3, along with the K_(i) for each respective compound, as measuredby the kinetic assay (last column). K_(i)'s for these compounds rangedfrom 7.7 nM for 3dp-4026 to 20.0 μM for 3dp-3811.

[0272] A stock human α-thrombin solution (1.56 mg/mL) from EnzymeResearch Labs was first diluted to 0.5 mg/mL (11 μM) with 50 mM Hepes,pH 7.5, 0.1 M NaCl (assay buffer, unless mentioned otherwise), andstored on ice. The five ligands (recrystallized solids characterized bymass spectrometry and NMR) were accurately weighed out to be 1.5 to 2.0mg and dissolved in 1.0 mL of 100% DMSO so that the concentration wasbetween 1.8 and 3.8 mM. A 96 well V-bottom Costar microplate was thenset up such that 100 μL of the 11 μM human α-thrombin solution waspipetted into wells A1 through A6. This was followed by the addition of2 μL of 3dp-3811 into well A2, 2 μL of 3dp-3959 into well A3, 2 μL of3dp-4077 into well A4, 2 μL of 3dp-4076 into well A5, 2 μL of 3 dp-4026into well A6, and 2 μL of 100% DMSO into control well A1. The contentswere mixed by repeated uptake and discharge using a 100 μL pipette tip.Finally, one drop of mineral oil (Sigma, St. Louis, Mo.) was added ontop of the wells to reduce evaporation of samples at elevatedtemperatures. The microplate was then placed on heating block 4 of aRoboCycler Gradient 96 Temperature Cycler (Stratagene, La Jolla,Calif.), set at 25° C., for 1 minute. The plate was then placed into aSPECTRAmax™ 250 spectrophotometer (set to 30° C.) and the absorbance at350 nm was measured for each sample. This reading served as the blank orreference from which all the other readings at higher temperatures werecompared. The assay was initiated by setting heating block 1 to 38° C.,programming the temperature cycler to move the microplate to heatingblock 1, and keeping the microplate there for 3 minutes. Following theequilibration at 38° C., the plate was moved to the 25° C. block (Block4) for 30 seconds, inserted in the spectrophotometer, and absorbance wasread at 350 nm. The microplate was then put back into the temperaturecycler and was moved to heating block 2, which had been pre-equilibratedat 40° C. After 3 minutes at 40° C., the plate was returned to 25° C.(on block 4) for 30 seconds, and was returned to the spectrophotometerfor a measurement of absorbance at 350 nm. This process was repeated 18more times until the temperature had been raised to 76° C. in 2° C.increments. After subtraction of the blank absorbance (A₃₅₀ at 25° C.),turbidity, reflected in the absorbance value, was plotted as a functionof temperature. The thermal denaturation curves for this experiment areshown in FIG. 1.

[0273] The control (in well A1), which contained only 11 μM humanα-thrombin in 2% DMSO, was found to undergo a thermal denaturationtransition starting at ˜50° C., as reflected in the large increase inA₃₅₀. The midpoint in this transition was observed to be ˜55° C. Thisresult was consistent with differential scanning calorimetricmeasurements for bovine prothrombin 1, which revealed a denaturationtransition at T_(m)=58° C. (Lentz, B. R. et al., Biochemistry33:5460-5468 (1994)). The thermal denaturation curves for all of thetested inhibitor compounds displayed a shift in the transition towardshigher temperatures. 3dp-4026 showed the largest shift in T_(m): ˜9° C.This result is consistent with the fact that, among the compoundstested, 3dp-4026 exhibited the greatest binding affinity; as judged bykinetic measurements with succinyl-Ala-Ala-Pro-Arg-p-nitroanilide assubstrate. Indeed, the rank order of shifts in T_(m), shown in FIG. 1,paralleled the order of binding affinity as measured by conventionalenzymology. These results indicate that by simply observing the shift inT_(m) for a series of compounds relative to the control, one can easilyand correctly rank a series of compounds in increasing order of bindingaffinity to the protein of interest

[0274] It was possible to take the microplate thermal shift assay onestep further and estimate the binding affinity of each ligand at T_(m).This was done by substituting T₀, T_(m), ΔH_(u) and ΔC_(pu) intoequation (1). If ΔH _(u) and ΔC_(pu) cannot be measured because acalorimetric device is not available, one can make educated guesses atΔH_(u) and ΔC_(pu) for the therapeutic target. In the case of humanα-thrombin, it was possible to use ΔH_(u)=200.0 kcal/mol, a valuemeasured for the closely related protein bovine prothrombin 1 (Lentz, B.R. et al., Biochemistry 33:5460-5468 (1994)). A value of ΔC_(pu)=2.0kcal/mol-° K was used to calculate K_(L) at T_(m) since similar proteinsof this size have been shown to yield similar values. The bindingaffinities at T_(m) of the five test ligands closely paralleled theK_(i)'s measured with a spectrophotometric substrate (Table 3). TABLE 3Microplate Thermal Shift Assay for Ligands Binding to the Active Site ofHuman α-thrombin. Turbidity as an Experimental Signal. [Li- K_(d) atK_(d) at K_(i) Protein/ gand] T_(m) ΔT_(m) T_(m) ^(a) 310° K^(b) (310°K)^(c) Ligand (μM) (° K) (° K) (nM) (nM) (nM) Thrombin (TH) none 327.150.0 TH/3dp-3811 37 328.15 1.0 14400 5880 2000 TH/3dp-3959 76 332.15 5.0660 224 250 TH/3dp-4077 48 333.15 6.0 160 51.7 46 TH/3dp-4076 60 334.157.0 76.3 23.6 26 TH/3dp-4026 67 336.15 9.0 12.3 3.5 7.7

EXAMPLE 2 Ranking Ligands that Bind to the Heparin Binding Site of Humanα-Thrombin

[0275] Assays for ligands that bind to the heparin binding site of humanα-thrombin are more difficult to perform than assays for ligands thatbind to the active site of human α-thrombin. At the heparin bindingsite, no substrate is hydrolyzed, so no spectrophotometric signal can beamplified for instrumental detection. Heparin activity is usuallyestimated in biological clotting time assays. Alternatively, heparinbinding affinity for human α-thrombin can be determined I 0 bylaboriously conducting 15 to 20 single point assays, in which theconcentration of low MW heparin is varied over two logs, and monitoringthe quenching of the fluorescent probe, p-aminobenzamidine, bound to theactive site of human α-thrombin (Olson, S. T. et al., J. Biol. Chem.266:6342-6352 (1991)). Thus, heparin binding to human α-thrombinrepresents the kind of challenge encountered with the vast majority ofnon-enzyme receptor/ligand binding events, which are commonly observedfor hormone/receptor interactions, repressor/DNA interactions,neurotransmitter/receptor interactions, etc. Several heparin-likesulfated oligosaccharides and sulfated naphthalene compounds wereassayed by the microplate thermal shift assay. Using the thermal shiftassay, it was possible to use a single compound per well to quickly rankthe compounds in order of increasing binding affinity, with K_(d)'sranging over three orders of magnitude (see Table 4). Like theexperiment in Example 1, the thermal shift assay results agreed closelywith the results obtained through an alternative method, which requireda series of laborious (15 to 20 single determinations) fluorescencequench assays over a wide range of concentrations of low MW heparin(Olson, S. T. et al., J. Biol. Chem. 266:6342-6352 (1991)). Theseresults confirm that by simply observing the shift in T_(m) for a seriesof compounds, relative to the control, one can easily and correctly ranka series of compounds in increasing order of binding affinity for theprotein of interest.

[0276] A search of the literature did not locate alternatively measuredbinding results for the other ligands, which may attest to thedifficulty of these experiments. However, the literature did reveal thatpentosan polysulfate (PSO₄) (Sigma, St. Louis, Mo.), dextran SO₄ (Sigma,St. Louis Mo.), and suramin (CalBiochem, LaJolla, Calif.) have beenobserved to have anticoagulant properties. Indeed, pentosan polysulfateand suramin were tested previously in clinical trials foranti-angiogenic activity, but were discounted due to toxic effects, manyof which were described as coagulation anomalies (Pluda, J. M. et al.,J. Natl. Cancer Inst. 85:1585-1592 (1993); Stein, C. A., Cancer Res.53:2239-2249 (1993)). The affinities of pentosan PSO₄ and suramin atT_(m), as measured by the thermal shift assay, were found to be 7-foldand 5700-fold higher, respectively, than the affinity of heparin 5000(Table 4). These results suggested that these ligands may alter clottingrates by interfering with the heparin mediated binding of humanα-thrombin to anti-thrombin III (AT III), a protein co-factor for humanα-thrombin activity.

[0277] The results in Table 4 revealed another advantage of themicroplate thermal shift assay for screening compound libraries: theprocess is blind and unbiased in the sense that it detects ligandbinding regardless of whether it is at the active site, an allostericcofactor binding site, or at a protein subunit interface. The ability todetect ligands that bind with high affinity to sites outside an enzyme'sactive site will greatly facilitate discovery of lead molecules. TABLE 4Microplate Thermal Shift Assay for Ligands Binding to the HeparinBinding Site of Human α-thrombin. Turbidity as an Experimental Signal.K_(d) at K_(i) [Li- 298° K^(b) (298° K) Protein/ gand] T_(m) ΔT_(m)K_(d) at T_(m) ^(a) (nM) (nM) Ligand (μM) (° K) (° K) (nM) ObservedLiterature Thrombin none 329.15 0.0 (TH) TH/Heparan 61 329.65 0.5 38,3007,570 — SO₄ TH/Heparin 50 330.15 1.0 19,700 3,810 — 3000 TH/Heparin 44330.15 1.0 17,200 3,490 5,400^(c) 5000 TH/Pentosan 40 332.15 3.0 2,425427 — PSO₄ TH/Dextran 48 336.15 7.0 68.8 10.1 — SO₄ TH/Suramin 102340.15 11.0 3.02 0.37 — #0.1 M NaCl (3-fold dilution). All ligands weredissolved in the same buffer.

EXAMPLE 3 Ranking aFGF Ligands

[0278] The second therapeutic receptor tested in the microplate thermalshift assay was acidic fibroblast growth factor (aFGF), a growth factorthat plays a key role in angiogenesis (Folkman, J. et al., J. Biol.Chem. 267:10931-10934 (1992)). A synthetic gene for this protein waspurchased from R&D Systems (Minneapolis, Minn.), and was cloned andexpressed in E. coli using methods similar to those described for basicfibroblast growth factor (bFGF) (Thompson, L. D. et al., Biochemistry33:3831-3840 (1994); Pantoliano, M. W. et al., Biochemistry33:10229-10248 (1994); Springer, B. A. et al., J. Biol. Chem.269:26879-26884 (1994)). Recombinant aFGF was then purified byheparin-sepharose affinity chromatography as described (Thompson, L. D.et al., Biochemistry 33:3831-3840 (1994)). aFGF is also known to bindheparin/heparan, which is a cofactor for mitogenic activity.Heparin-like molecules, such as pentosan PSO₄ and suramin, inhibit thegrowth factor's biological activity. A microplate thermal assay of thesecompounds was set up in a way similar to that described above for humanα-thrombin. The change in turbidity, as a function of temperature, foreach of the ligands suramin, heparin 5000, and pentosan PSO₄, is shownin FIG. 2. The results are summarized in Table 5. The affinity constantscovered a fairly broad range of binding affinities, with pentosan PSO₄showing the highest affinity. The order of ligand binding affinity ofpentosan PSO₄, heparin 5000 and suramin paralleled that found for bFGF,as measured using isothermal titrating calorimetry (Pantoliano, M. W. etal., Biochemistry 33:10229-10248 (1994)). The lack of alternativelymeasured binding affinities for these compounds probably attests to thedifficulty of making these measurements using assays which do notmonitor physical, temperature-dependent changes.

[0279] The results in Table 5 are consistent with the results in Tables3 and 4. Simply observing the shift in T_(m) for a series of compoundsrelative to the control, one can easily and correctly rank a series ofcompounds in increasing order of binding affinity to the protein ofinterest. TABLE 5 Microplate Thermal Shift Assay for Ligands Binding toaFGF. Turbidity as an Experimental Signal. K_(d) at K_(i) 298° K^(b)(298° K)^(c) Protein/ [Ligand] T_(m) ΔT_(m) K_(d) at T_(m) ^(a) (nM)(nM) Ligand (μM) (° K) (° K) (nM) Observed Literature aFGF none 317.150.0 aFGF/EEEEE 50 317.15 0.0 >50,000 — aFGF/Dermatan SO₄ 50 318.15 1.037,000 12,700 — aFGF/EEEEEEEE 50 322.15 5.0 10,076 3,040 — aFGF/β-CD 14SO₄ 47 329.15 12.0 1055 213 1500 aFGF/suramin 200 330.15 13.0 3220 622aFGF/Heparin 5000 50 331.15 14.0 576 106 470 aFGF/Heparan SO₄ 61 333.1516.0 357 60 — aFGF/Pentosan PSO₄ 100 336.15 19.0 208 31 88 #AmericanMaize Products Co. (Hammond, IN). The aFGF was diluted to 0.25 mg/mL in50 mM Hepes, pH 7.5, 0.1 M NaCl. All ligands were dissolved in the samebuffer.

EXAMPLE 4 Ranking bFGF Ligands

[0280] The microplate thermal shift assay was used to assess ligands forbinding to the heparin binding site of basic fibroblast growth factor(bFGF). The gene for bFGF was purchased from R&D Systems and was clonedand expressed in E. coli as previously described (Thompson, L. D. etal., Biochemistry 33:3831-3840 (1994); Pantoliano, M. W. et al.,Biochemistry 33:10229-10248 (1994); Springer, B. A. et al., J. Biol.Chem. 269:26879-26884 (1994)). It was found that pentosan PSO₄ andsuramin bound to bFGF with binding affinities of 55 nM and 3.5 μM,respectively. This result for PSO₄ compared very well with the affinityof 88 nM observed for PSO₄ binding to bFGF, as determined by isothermaltitrating calorimetry.

EXAMPLE 5 Ranking Human α-Thrombin Ligands Using Fluorescence Emission

[0281] Because fluorescence measurements are more sensitive thanabsorbance measurements, a fluorescence thermal shift assay was used toassess ligand binding to human α-thrombin. The fluorescence emissionspectra of many fluorophores are sensitive to the polarity of theirsurrounding environment and therefore are effective probes of phasetransitions for proteins (i.e., from the native to the unfolded phase).The most studied example of these environment dependent fluorophores is8-anilinonaphthalene-1-sulfonate (1,8-ANS), for which it has beenobserved that the emission spectrum shifts to shorter wavelengths (blueshifts) as the solvent polarity decreases. These blue shifts are usuallyaccompanied by an increase in the fluorescence quantum yield of thefluorophore. In the case of ANS, the quantum yield is 0.002 in water andincreases to 0.4 when ANS is bound to serum albumin.

[0282] ANS was used as a fluorescence probe molecule to monitor proteindenaturation. In the fluorescence assay, the final concentration ofhuman α-thrombin was 0.5 μM, which is 20-fold more dilute than theconcentrations used in the turbidity assays. This concentration of humanα-thrombin is in the range used for the kinetic screening assays.

[0283] ANS was excited with light at a wavelength of 360 nm. Thefluorescence emission was measured at 460 nm using a CytoFluor IIfluorescence microplate reader (PerSeptive Biosystems, Framingham,Mass.). The temperature was ramped up as described above for theturbidity assays (see Example 1). The plot of fluorescence as a functionof temperature is shown in FIG. 3 for human α-thrombin alone, and forthe 3dp-4026/human α-thrombin complex. The denaturation transition forhuman α-thrombin was clearly observed at 57° C., a temperature which isonly slightly higher than that observed in the turbidity experiment. Theresult from the fluorescence assay is, nonetheless, in close agreementwith the T_(m) of 58° C. observed for prothrombin 1 from differentialscanning calorimetry experiments. Importantly, 3dp-4026 (at 67 μM) wasfound to shift the denaturation transition to ˜66° C. to give a shift inT_(m) of 9° C., which is identical to that found using turbidity as thedetection signal (Table 3).

[0284] The results in FIG. 3 and Table 4 illustrate several importantpoints. First, at least a 20-fold increase in sensitivity can be gainedby switching from an absorbance to a fluorescence emission detectionsystem. This can be critical for those receptor proteins for whichsupplies are limited.

[0285] Second, in the fluorescence assays, the denaturation transitionsignal is much cleaner than the signal in the turbidity assays. In theturbidity assays, higher concentrations of protein led to precipitationof denatured protein. Precipitated protein contributed to the noisysignal.

[0286] Third, shifts in T_(m) measurements from the microplate thermalshift assays are reproducible from one detection system to another.

EXAMPLE 6 Ranking Ligands to the D(II) Domain of FGFR1

[0287] The microplate thermal-shift assay was employed to test thebinding of heparin 5000 and pentosan PSO₄ to the known heparin bindingsite in the D(II) domain of fibroblast growth factor receptor 1 (FGFR1).D(II) FGFR1 is a 124 residue domain which is responsible for most of thefree energy of binding for bFGF. D(II) FGFR1 was cloned and expressed inE. coli. Recombinant D(II) FGFR1 was renatured from inclusion bodiesessentially as described (Wetmore, D. R. et al., Proc. Soc. Mtg., SanDiego, Calif. (1994)), except that a hexa-histidine tag was included atthe N-terminus to facilitate recovery by affinity chromatography on aNi²⁺ chelate column (Janknecht, R. et al., Natl. Acad. Sci. USA88:8972-8976 (1991)). D(II) FGFR1 was further purified on aheparin-sepharose column (Kan, M. et al., Science 259:1918-1921 (1993);Pantoliano, M. W. et al., Biochemistry 33:10229-10248 (1994)). Puritywas >95%, as judged by SDS-PAGE. The D(II) FGFR1 protein wasconcentrated to 12 mg/mL (˜1 mM) and stored at 4° C.

[0288] The D(II) FGFR1 protein was dissolved in an ANS solution to aconcentration of 1.0 mg/mL (70 μM). The quantum yield for ANS bound tothe denatured form of D(II) FGFR1 was lower than the quantum yield forANS bound to human α-thrombin. Because ANS fluorescence is veryenvironment dependent (see Lakowicz, I. R., Principles of FluorescenceSpectroscopy, Plenum Press, New York (1983)), the quantum yield observedfor the denaturation of different proteins will vary. For D(II) FGFR1,the signal for the turbidity version of the assay, however, was nearlyundetectable. Despite the decreased sensitivity for D(II) FGFR1, ANSrescued this system for the microplate assay. A similar result wasobtained for Factor Xa, except that the fluorescence quantum yield forANS bound to denatured Factor Xa was almost as good as it was for humanα-thrombin. It was found that the fluorescence quantum yield for ANSbound to denatured bFGF was as high as the quantum yield for ANS bindingto human α-thrombin.

[0289] The results of D(II) FGFR1 binding experiments, as determined bythe microplate thermal shift assay, are shown in FIG. 4 and Table 6. Aswas previously demonstrated for all of the other receptor proteinsdescribed above, the microplate thermal shift assay facilitated theranking of ligand binding affinities for D(II) FGFR1. TABLE 6 MicroplateThermal Shift Assay for Ligands Binding to D(II) FGFR1. FluorescenceEmission as an Experimental Signal. K_(d) at K_(d) K_(d) at 298° K^(b)(298° K)^(c) [Ligand] T_(m) ΔT_(m) T_(m) ^(a) (μM) (μM) Protein/Ligand(μM) (° K) (° K) (μM) Observed Literature D(II) FGFR1 none 312.8 0.0D(II) FGFR1/ 150 317.9 5.1 30.0 13.6 85.3 Heparin 5000 D(11) FGFR1/ 156319.4 6.6 19.1 4.9 10.9 Pentosan PSO₄

EXAMPLE 7 Microplate Thermal Shift Assay of Factor D

[0290] In order to further demonstrate the cross target utility of themicroplate thermal shift assay, another enzyme, Factor D, was tested forits ability to undergo thermal unfolding transitions. Factor D is anessential serine protease involved in the activation of the alternativepathway of the complement system, the major effector system of the hostdefense against invading pathogens. Factor D was purified from the urineof a patient with Fanconi's syndrome (Narayana et al., J. Mol. Biol.235:695-708 (1994)) and diluted to 4 μM in assay buffer (50 mM Hepes, pH7.5, 0.1 M NaCl). The assay volume was 10 μL and the concentration of1,8-ANS was 100 μM. The experiment was carried out using 15 μL roundbottom dimple plates (an 8×12 well array). The protein was heated in two30 degree increments between 42° C. to 62° C., using a Robocycler™temperature cycler. After each heating step, and prior to fluorescencescanning using the CytoFluor II™ fluorescence plate reader the samplewas cooled to 25° C. (see Example 1). The non-linear least squares curvefitting and other data analysis were performed as described for FIG. 3.The results of the microplate thermal shift assay of Factor D is shownin FIG. 5 and reveal a thermal unfolding transition that occurs near 324K (51° C.) for the unliganded form of the protein. No reversible ligandsof significant affinity are known for Factor D. The results in FIG. 5show that the microplate thermal shift assay can be used to screen alibrary of compounds for Factor D ligands. The results in FIG. 5 alsoshow that the microplate thermal shift assay is generally applicable toany target molecule.

EXAMPLE 8 Microplate Thermal Shift Assay of Factor Xa

[0291] Human Factor Xa, a key enzyme in the blood clotting coagulationpathway, was chosen as yet another test of the cross target utility ofthe microplate thermal shift assay. Factor Xa was purchased from Enzymeresearch Labs (South Bend, Ind.) and diluted to 1.4 μM in assay buffer(50 mM Hepes, pH 7.5, 0.1 M NaCl). The assay volume was 100 μL and theconcentration of 1,8-ANS was 100 μM. The protein was heated in twodegree increments between 50° C. to 80° C. using a Robocycler™temperature cycler. After each heating step, prior to fluorescencescanning using the CytoFluor II™ fluorescence plate reader, the samplewas cooled to 25° C. (see Example 1). The results of a microplatethermal shift assay of Factor Xa is shown in FIG. 6. A thermal unfoldingtransition was observed at 338K (65° C.). Data analysis was described asdescribed for FIG. 3. The results in FIG. 6 show that the microplatethermal shift assay of protein stability is generally applicable to anytarget molecule.

EXAMPLE 9 Miniaturization of the Microplate Thermal Shift Assay ofLigands Binding to Human α-Thrombin

[0292] A miniaturzed form of the microplate thermal shift assay wasdeveloped to minimize the amount of valuable therapeutic protein andligands required for the assay. In the first attempt at decreasing theassay volume, the assay volume was decreased from 100 μL to 50 μLwithout adversely affecting the fluorescent signal. When the assayvolume was reduced further by a factor of ten, to 5 μL, favorableresults were obtained for human α-thrombin. As shown in FIG. 7, thehuman α-thrombin unfolding transition could be easily observed at itsusual T_(m). More importantly, an active site inhibitor was observed toshift the T_(m) of the unfolding transition by 8.3° K to yield anestimate of the K_(d) of 15 nM at the T_(m). The K_(a) at T_(m) wascalculated using the relationship: $\begin{matrix}{K_{L}^{T_{m}} = \frac{\begin{matrix}{\exp \{ {{- {\frac{\Delta \quad H_{u}^{T_{0}}}{R}\lbrack {\frac{1}{T_{m}} - \frac{1}{T_{0}}} \rbrack}} +} } \\ \quad {\frac{\Delta \quad C_{pu}}{R}\lbrack {{\ln ( \frac{T_{m}}{T_{0}} )} + \frac{T_{0}}{T_{m}} - 1} \rbrack} \}\end{matrix}}{\lbrack L_{T_{m}} \rbrack}} & ( {{equation}\quad 1} )\end{matrix}$

[0293] where K_(L) ^(T) ^(_(m)) =K_(a) at T_(m) (ligand associateconstant at T_(m))

[0294] T_(m)=332.2° K (midpoint of the unfolding transition in theabsence of a ligand)

[0295] T₀=323.9° K

[0296] ΔH_(u) ^(T) ^(_(m)) =200.0 kcal/mol (enthalpy of unfolding forpre thrombin observed by Lentz et al., 1994)

[0297] ΔC_(pu)=2.0 kcal/mol (estimated change in heat capacity ofunfolding for human α-thrombin)

[0298] L_(T) _(m) =50.0 μM

[0299] The Kd at temperatures near 25 or 37° C. will be of higheraffinity if the enthalpy of binding, ΔH_(b), is negative for thisligand. Using a spectrophotometric assay, an apparent K_(i) ofapproximately 8 nM was observed at 37° C. (310° K).

[0300] The measurements shown in FIG. 7 were obtained using theCytoFluor II fluorescence plate reader (PerSeptive Biosystems,Framingham, Mass.). In the experiment, the excitation wavelength oflight was 360 nm and the emission was measured at 460 nm. Themicroplates employed for this miniaturized assay were either theconventional polycarbonate V-bottom 96 well plate (Stratagene, orCostar) or polycarbonate plates that contain 15 μL dimples in an 8×12array (Costar plate lids). In the reaction, the concentration of humanα-thrombin was μM in assay buffer (50 mM Hepes, pH 7.5, 0.1 M NaCl). Theassay volume was 5 μL and the concentration of 1,8-ANS was 100 μM. Theprotein was heated in two degree increments between 44° C. to 64°C.using a Robocycler™ temperature cycler. After each heating step, andprior to fluorescence scanning using the CytoFluor II™ fluorescenceplate reader the sample was cooled to 25° C. for 30 seconds (see Example1). The non-linear least squares curve fitting and other data analysiswere performed as described for FIG. 3.

EXAMPLE 10 Miniaturization of the Microplate Thermal Shift Assay ofLigands Binding to D(II) FGFR1

[0301] Recombinant D(II) FGFR1 was purified from inclusion bodies andpurified by affinity chromatography on heparin sepharose. A stocksolution of D(II) FGFR1 (15 mg/ml; 1.1 mM) was diluted to 50 μM in assaybuffer (50 mM Hepes, pH 7.5, 0.1 M NaCl). The assay volume was 10 μL andthe concentration of 1,8-ANS was 250 μM. The unfolding transition in theabsence of ligands was found to be about 312 K (39° C.) as shown in FIG.8. In the presence of the heparin mimic aprosulate (300 uM), theunfoding transition was observed to increase by about 8 K to about 320K. Using this temperature midpoint T_(m), it is possible to estimate thebinding affinity of aprosulate to D(II)FGFR1 to be about 18 μM at theT_(m) (Table 6). These results demonstrate the ability of the microplatethermal shift assay to estimate ligand binding affinity to a non-enzymetarget molecule.

EXAMPLE 11 Miniaturization of the Microplate Thermal Shift Assay ofUrokinase

[0302] Another target molecule analyzed was human urokinase-typeplasminogen activator (u-PA). U-PA enzymatically converts plasminogeninto the active protease plasmin. U-PA is involved in tissue remodeling,cellular migration and metastases. The gene for u-PA was obtained fromATCC (Rockville, Md.) and modified to appropriately express activeenzyme in E. coli. u-PA was cloned, overexpressed in E. coli, andpurified using procedures similar to those described by Winkler et al.(Biochemistry 25:4041-4045 (1986)). The last step of u-PA purificationwas performed in the presence of the active site inhibitorglu-gly-arg-chloromethylketone (CMK) and hence the u-PA utilized forthis experiment was the CMK-u-PA complex. The experiment was performedin the miniaturized format in 5 μL well volume. One μL of concentratedCMK-u-PA (13 g/L, 371.4 μM) was added to 4 μL of 62.5 mM MOPS, pH 7, 125mM NaCl, and 250 μM 1,8-ANS, in multiple wells of a 96-wellpolycarbonate V-bottom microtiter plate. A thermal denaturation curvewas generated as previously described for thrombin, aFGF, D(II)FGFR1,Factor D, and Factor Xa, by incremental heating of the microplatefollowed by a fluorescence reading after each temperature increase.Analysis and non-linear least squares fitting of the data for thisexperiment show that the T_(m) for CMK-u-PA under these conditions is81° C., which is considerably higher than that seen for thrombin, aFGF,D(II)FGFR1, Factor D, and Factor Xa (55,44,40, 51, 55, and 65° C.,respectively). This experiment demonstrates the utility of the currentinvention in determining the T_(m) for relatively thermostable proteinsor proteins stabilized by the high affinity binding of ligand(s) andfurther demonstrates the ability to perform such an experiment in aminiaturized format.

EXAMPLE 12 Further Miniaturization of the Microplate Thermal Shift Assayof Human α-Thrombin

[0303] A stock thrombin solution was diluted to 1 μM in 50 mM Hepes, pH7.5, 0.1 M NaCl and 100 μM 1,8-ANS. An electronic multi-channel pipettorwas used to dispense either 2 μL or 5 μL of diluted thrombin solutioninto wells of a 96-well polycarbonate microtiter plat. The plate wassubjected to 3 minutes of heating in a thermal block capable ofestablishing a temperature gradient across the microplate, followed by30 seconds cooling to 25° C., and subsequent reading in the CytoFluor IIfluorescence plate reader. Data were analyzed by non-linear leastsquares fitting and plotted as shown in FIGS. 10 and 11. Each curverepresents a replicate experiment. Standard deviations for T_(m)determinations were very good for experiments utilizing either 5 μL or 2μL volumes (+/−1.73 and +/−0.90 K, respectively), demonstrating theability of the current invention to operate at very low volumes. Infact, the volume which one could employ in the current invention seemsto be limited only by the technology available to dispense small volumesaccurately.

[0304] The assay volume was reduced to 2 μL, as shown for humanα-thrombin (1.0 μM) in FIG. 11. Reproducible pipetting of 2 μL in a 96well array requires the employment of specialized pipetting tools suchas the multi-channel pipettor available from Matrix Technologies Corp.(Lowell, Mass.) which has ±2.0% or 0.15 μL precision and ±2.5% or 0.15μL accuracy for volumes 0.5 to 12.5 μL.

EXAMPLE 13 Single Temperature Mode of the Microplate Thermal Shift Assay

[0305] Results of a single temperature assay are shown in FIG. 12. Thecompounds 3DP-3811, 3DP-3959, 3DP-4076, and 3DP-4660 bind to the activesite of human α-thrombin. The K_(i)'s (enzymatically determined) ofthese four compounds for human α-thrombin are of 20,000 nM, 250 nM, 25nM, and 8 nM, respectively. Each of these four compounds wereequilibrated with human α-thrombin in separate 5 μl assay volumes in a96 well plate. The final ligand concentration was 50 μM.

[0306] For the ligands that bind to human α-thrombin with higheraffinity, low levels of fluorescence emission were observed, relative tothe control reaction (human α-thrombin alone) at 55° C. The result forthe sample containing the weakly binding ligand 3DP-3811 was littledifferent from the result obtained for the control sample. The decreasein fluorescence emission for 3DP-4076 was not as large as expected,given its high affinity (K_(i) of 25 nM) for human α-thrombin. Thisresult could be due in part to the lower solubility of chloride salts ofthis compound.

[0307] The data in FIG. 12 clearly demonstrate the utility of the singletemperature embodiment of the microplate thermal shift assay for quicklyidentifying ligands with binding affinities (K_(d)'s) of 250 nM orbetter when the ligand concentration is 50 μM.

EXAMPLE 14 Microplate Thermal Shift Assay of Intrinsic ProteinTryptophan Fluorescence Emission

[0308] The intrinsic Trp fluorescence of human α-thrombin was assayed ina microplate thermal shift assay. 100 μL samples contained 2 μM humanα-thrombin. The samples were exposed to light from a Xenon-Arc lamp at280 nm. Emission was detected at 350 nm using the BioLumin 960(Molecular Dynamics). Temperature cycling, between 44° C. and 66° C.,was performed as described in previous examples. The results of theassay are shown in FIGS. 13 and 14. A small increase in fluorescenceemission was observed at 350 nm with increasing temperature. However,this increase in fluorescence emission was barely detectable above thelevel of fluorescence in the blank wells that contained no protein (FIG.13). Subtracting an average blank improved the signal to noise ratio(FIG. 14), but the observed unfolding transition was different from thattypically observed in assays employing 1,8-ANS. In contrast to thetransition observed using 1,8-ANS, the transition in FIG. 14 appearsbroader and has a midpoint temperature T_(m) at 334.4±5.1° K, some fivedegrees higher than the T_(m) observed for human α-thrombin in assaysperformed with 1,8-ANS.

EXAMPLE 15 Assay of Multi-Ligand Binding Interactions

[0309] As previously demonstrated, the thermal shift assay can be usedfor the screening of ligands for binding to single sites on targetproteins. In light of the underlying physical principles upon which themicroplate thermal shift assay is based, the near additivity of the freeenergy of ligand binding and protein unfolding, it is possible to employthe microplate thermal shift assay for analyzing multi-ligand bindinginteractions with a target protein. If the free energy of binding ofdifferent ligands binding to the same protein are nearly additive, thenone can analyze multi-ligand binding systems, whether the ligands bindin a cooperative (positive) fashion or a non-cooperative (negative)fashion.

[0310] Multiple ligand binding to human α-thrombin was assayed in amicroplate thermal shift assay. Human α-thrombin it has at least fourdifferent ligand binding sites: (1) the catalytic binding site; (2) thefibrin binding site (exosite I); (3) the heparin binding site (exositeII); and (4) the Na⁺ binding site, located ˜15 Å from the catalyticsite. First, independent binding of three individual ligands wasassayed: 3DP-4660, Hirugen (hirudin 53-64) (Bachem), and heparin 5000(CalBiochem). These ligands bind to the catalytic site, the fibrinbinding site and the heparin binding site, respectively.

[0311] A stock thrombin solution was diluted to 1 μM in 50 mM Hepes, pH7.5, 0.1 M NaCl, 1 mM CaCl₂, and 100 μM 1,8-ANS. Each thrombin ligandwas included singly and in various combinations to 1 μM thrombinsolutions at final concentrations of 50 μM each, except for heparin5000, which was 209 μM. 100 μL of thrombin or thrombin/ligand(s)solution was dispensed into wells of a 96-well V-bottom polycarbonatemicrotiter plate. The plate was subjected to 3 minutes of heating in athermal block capable of establishing a temperature gradient across themicroplate, followed by 30 seconds cooling at 25° C., and subsequentreading in a fluorescence plate reader. Data were analyzed by non-linearleast squares fitting.

[0312] The results of these individual binding reactions are shown inFIGS. 15 and 16. The rank order of binding affinity was3DP-4660 >Hirugen >heparin 5000, corresponding to K_(d) values of 15 nM,185 nM and 3434 nM, respectively, for the ligands binding at each T_(m)(see Equation (4)).

[0313] The results reveal thermal unfolding shifts that are slightlysmaller than would be expected if the free energies of binding werefully additive. For example, Hirugen alone displays a ΔT_(m) of 5.8° C.,and 3DP-4660 alone displays a ΔT_(m) of 7.7° C. In combination, however,Hirugen and 3DP-4660 display a ΔT_(m) of 12.2° C. This result means thatthe binding affinity of one or both ligands is diminished when bothligands are bound, and is an example of negative cooperativity inbinding between the fibrin and catalytic binding sites. Such anegatively cooperative effect is consistent with the human α-thrombinliterature, in which the kinetics of hydrolysis of various chromogenicsubstrates were found to depend upon ligands binding to exosite I.Indeed, a 60% decrease in K_(m) for the hydrolysis ofD-phenylalanylpipecolyl arginyl-p-nitroanilide was observed when Hirugenwas present (Dennis et al., Eur. J. Biochem. 188:61-66 (1990)).Moreover, there is also structural evidence for cooperativity betweenthe catalytic site and exosite I. A comparison of the isomorphousstructures of human α-thrombin bound to PPACK (a human α-thrombincatalytic site inhibitor) and Hirugen revealed conformational changesthat occur at the active site as a result of Hirugen binding at theexosite I (Vijayalakshmi et al., Protein Science 3:2254-2271 (1994)).Thus, in the microplate thermal shift assay, the apparent cooperativityobserved between the catalytic center and the exosite I is consistentwith functional and structural data in the literature.

[0314] Similarly, when the binding of all three ligands was assayed, aΔT_(m) of 12.9° C. was observed (FIG. 16). If the free energies ofbinding were fully additive, one would expect to observe a ΔT_(m) of17.7° C. The observed result means that further negative cooperativityoccurs via ligand binding at all three protein binding sites. Thisresult is consistent with the literature. In a ternary complex withheparin and fibrin monomer, human α-thrombin has decreased activitytoward tri-peptide chromogenic substrates and pro-thrombin (Hogg &Jackson, J. Biol. Chem. 265:248-255 (1990)), and markedly reducedreactivity with anti-thrombin (Hogg & Jackson, Proc. Natl. Acad. Sci.USA 86:3619-3623 (1989)). Also, recent observations indicate thatternary complexes also form in plasma and markedly compromise heparinanticoagulant activity (Hotchkiss et al., Blood 84:498-503 (1994)). Asummary of these multi-ligand binding results is shown in Table 7.

[0315] The results in FIG. 15, FIG. 16, Table 7 illustrate the followingadvantages of using the microplate thermal shift assay to performmulti-variable analyses. First, the same microplate thermal shift assaycan be used to simultaneously detect the binding of multiple ligands atmultiple binding sites in a target protein. Second, the microplatethermal shift assay can be used to detect the same ligand binding to twoor more sites in a therapeutic target. Third, the microplate thermalshift assay affords the detection of cooperativity in ligand binding.Information about ligand binding cooperativity can be collected andanalyzed very quickly. Thus, multi-ligand binding experiments that wouldtake months to perform using alternative technologies take only hours toperform using the microplate thermal shift assay. TABLE 7 Microplatethermal shift assay for Ligands Binding to the Active Site, Exosite, andHeparin Binding Site of Human α-thrombin K_(d) at [Ligand] T_(m) Δ T_(m)K_(d) at T_(m) ^(a) 298°^(b) Protein/Ligand (μM) (° K) (° K) (nM) (nM)Thrombin (TH) none 323.75 0.0 TH/Heparin 5000 200 327.95 4.2 3434 470TH/Hirudin 53-65 50 329.52 5.8 185 23 TH/3dp-4660 50 331.40 7.7 29 3TH/Heparin 5000 200 327.95 TH/Hep./Hir. 50 330.57 2.6 4254 478TH/Heparin 5000 200 327.95 TH/Hep.3dp 4660 50 333.20 5.3 350 32TH/Hirudin 53-65 50 329.52 TH/Hir./Hep. 200 330.57 1.1 75422 8467TH/Hirudin 53-65 50 329.52 TH/Hir.3dp-4660 50 335.97 6.5 117 9TH/3dp-4660 50 331.40 TH/3dp-4660/Hep 200 333.20 1.8 38205 351TH/3dp-4660 50 331.40 TH/3dp-4660/Hir. 50 335.97 4.6 731 54

EXAMPLE 16 Screening Biochemical Conditions that Increase Humanα-Thrombin Stability

[0316] The microplate thermal shift assay was used, with four differentfluorophores, to simultaneously screen the effects of multiple pHvalues, sodium chloride concentrations, and reduction-oxidationcompounds on human α-thrombin stability. Thrombin solution was dilutedto 1 μM in 50 mM Hepes, pH 7.5, NaCl at either 0.1 M or 0.5 M, 10 mMEDTA, 10 mM CaCl₂, 10 mM dithiothreitol, 10 1 mM CaCl₂, and 100 μM1,8-ANS, 10% (v/v) glycerol, or 0.1% (w/v) polyethylene glycol (PEG)6000. Reaction volume was 100 μL.

[0317] The results of these multi-variable experiments are shown inFIGS. 17A-D and FIG. 18. FIGS. 17A-D summarize the stability datacollected in a single 96 well plate for human α-thrombin. In FIG. 17A,the fluorophore is 1,8-ANS. In FIG. 17B, the fluorophore is 2,6-ANS. InFIG. 17C, the fluorophore is 2,6-TNS. In FIG. 17D, the fluorophore isbis-ANS. The results in FIGS. 17A-D show a pH optimum of about 7.0 andan increase in stability with increasing NaCl concentration. A ΔT_(m) ofabout 12° C. was observed when the NaCl concentration was increased from0 to 0.5 M. FIG. 18 shows a stabilizing effect of 10% glycerol and adestabilizing effect of dithiothreitol. From FIGS. 17A-D and 18 isevident that the flourophores 1,8-ANS and 2,6-TNS are most effective inthe microplate thermal shift assay.

[0318] The stabilizing effect of NaCl is particularly interesting sincethere are recent reports in the literature of a weak Na⁺ binding site(K_(d) of 30±3 mM in 5 mM Tris buffer pH 8.0, 0.1% PEG, 25° C.)approximately 15 Å from the catalytic center of thrombin (Dang et al.,Nature Biotechnology 15:146-149 (1997)). Using equation (1), it ispossible to estimate the NaCl binding to be ˜6 mM near the T_(m) (53°C.) in 50 mM Hepes. pH 8.0 buffer (zero and 0.10 M NaCl).

[0319] The additional stabilization that occurs at a NaCl concentrationof greater than 0.10 M may come from additional Na⁺ and/or Cl⁻ bindingevents summed over the entire structure of human α-thrombin.Alternatively, the source of this further stabilization may come fromless specific salting out effect that is usually observed at 0.5 to 2 MNaCl and is due to the preferential hydration of proteins induced bysalts (Timasheff & Arakawa, In: Protein Structure, A Practical Approach,T. E. Creighton, ed., IRL Press, Oxford, UK (1989), pp. 331-354)).

[0320] The stabilizing effect of glycerol on proteins has beenattributed to a balance between the preferential exclusion of glycerol(i.e. preferential hydration of proteins) and the specific binding topolar regions on the surface of proteins (Timasheff & Arakawa, In:Protein Structure, A Practical Approach, T. E. Creighton, ed., IRLPress, Oxford, UK (1989), pp. 331-354)).

EXAMPLE 17 Screening Biochemical Conditions that Increase D(II) FGFReceptor 1 Stability

[0321] The microplate thermal shift assay was used to simultaneouslyscreen the effects of multiple biochemical conditions on D(II) FGFreceptor 1 stability. The assays were performed by mixing 1 μL of D(II)FGFR1 (from a 500 μM concentrated stock in 50 mM HEPES pH 7.5) with 4 μLof each biochemical condition in wells of a 96-well polycarbonatemicrotiter plate. Final protein concentration after mixing was 100 μMand final. 1,8-ANS concentration was 200 μM. Biochemical conditions weretested as follows: The pH's tested were 5 (Na acetate), 6 (MES), 7(MOPS), 8 (HEPES), and 9 (CHES), with final buffer concentrations of 50mM.

[0322] The salt concentrations tested were 0.1 or 0.5 M NaCl. Additiveswere tested in 50 mM MOPS, pH 7, 0.1 M NaCl, at final concentrations of1 mM (EDTA, dithiothreitol), 10 mM (CaCl₂, MgCl₂, MgSO₄, NiSO₄), 50 mM(arginine), 100 mM (NH₄)₂SO₄, LiSO₄, Na₂SO₄, ZnSO₄), 5% w/v(polyethylene glycol 6000), and 10% v/v glycerol.

[0323] Thermal denaturation profiles were generated as previouslydescribed for thrombin, aFGF, Factor D, and Factor Xa, by incrementalheating of the microplate followed by a fluorescence reading after eachtemperature increase. Data were analyzed by non-linear least squaresfitting as described previously.

[0324] The results of these multi-variable experiments are shown inFIGS. 19-24. As shown in FIG. 19, stability increased with increasingNaCl concentration. A ΔT_(m) of about 5° C. was observed as NaClconcentration was increased from 0.1 to 0.5 M. As shown in FIG. 20, bothMgSO₄ and arginine stabilized the protein. As shown in FIG. 21, 10%glycerol stabilized the protein. Further, salts of the Hofmeister seriessuch as Li₂SO₄, Na₂SO,₄ (NH₄)₂SO₄ and Mg₂SO₄ all had stabilizing effects(FIG. 21). As shown in FIG. 22, dithiothreitol destabililzed theprotein. These results are not very different form that of humanα-thrombin. As shown in FIG. 23, a pH optimum of about 8.0 was observed.The relative stabilizing effects of EDTA, CaCl₂, MgCl₂, MgSO₄, arginine,(NH₄)₂SO₄Li₂SO,₄Na₂SO,₄ glycerol, polyethylene glycol 6000, anddithiothreitol are shown in FIG. 24.

EXAMPLE 18 Screening Biochemical Conditions that Increase UrokinaseStability

[0325] The microplate thermal shift assay was used to simultaneouslyscreen the effects of multiple biochemical conditions on human urokinasestability. This experiment was performed by mixing 1 μL of urokinase(from a 371 5M concentrated stock in 20 mM Tris pH 8) with 4 μL of eachbiochemical condition in wells of a 96-well polycarbonate microtiterplate. Final protein concentration after mixing was 74 μM and final1,8-ANS concentration was 200 μM. Biochemical conditions were tested asfollows: The pH's tested were 5 (acetate), 6 (MES), 7 (MOPS), 8 (HEPES),and 9 (CHES) with final buffer concentrations of 50 mM. The saltconcentrations tested were 0.1 or 0.5 M NaCl. Glycerol was tested at 10%v/v in 50 mM MOPS, pH 7, 0.1 M NaCl.

[0326] Thermal denaturation profiles were generated as previouslydescribed for thrombin, aFGF, Factor D, D(II) FGFR1, and Factor Xa, byincremental heating of the microplate followed by a fluorescence readingafter each temperature increase. Data were analyzed by non-linear leastsquares fitting as described previously.

[0327] The results of these multi-variable experiments are shown in FIG.25. A pH optimum of about 7.0 was observed. Increasing concentrations ofsodium chloride stabilized the protein. 10% glycerol also stabilized theprotein. These results are consistent with the results reported in theliterature (Timasheff & Arakawa, In: Protein Structure, A PracticalApproach, T. E. Creighton, ed., IRL Press, Oxford, UK (1989), pp.331-354).

[0328]FIGS. 17-25 illustrate the advantage of using the microplatethermal shift assay to simultaneously screen for multi-variablebiochemical conditions that optimize protein stability. Using themethods and apparatus of the present invention, one can rapidly screenlarge arrays of biochemical conditions for conditions that influence thestability of proteins. Thus, the present invention can be used torapidly identify biochemical conditions that optimize proteinshelf-life.

EXAMPLE 19 Screening Biochemical Conditions that Facilitate ProteinFolding

[0329] Factorial experiments were performed to identify biochemicalconditions that increased the yield of correctly foldedHis₆-D(II)-FGFR1. His₆-D(II)-FGFR1 is recombinant D(II) FGF receptor 1protein, to which a polyhistidine tag is attached to the N-terminus. Theresults are summarized in Table 8. When the final guanidiniumhydrochloride concentration was 0.38 M, a refolded protein yield of13.5+0.2% was obtained at pH 8.0 and 0.5 M NaCl. This yield could beincreased to 15.5±0.3% if glycerol was present at 7% (v/v). A furtherincrease in His₆-D(II)-FGFRI refolding yield to about 18% was observedwhen the pH was increased to 8.9. In fact, increasing the pH from 8.0 to8.9 improved the yields in all experiments. These results demonstratethat a pH between 8 and 9, and 7% glycerol, are two important conditionsthat facilitate D(II)-FGFRI folding. Each of these conditions increasedthe folded protein yield by about 15 to 20% over the starting conditionsat pH 8.0 and 0.5 M NaCl.

[0330] Importantly, the effects of pH and glycerol appear to be nearlyadditive. The increased yield of refolded protein at pH 8.9 and 7%glycerol was found to be 17.8%, 32% higher than the yield obtained at apH 8.0 and 0.5 M NaCl (13.5±0.2% yield). The near additivity ofrefolding determinants has important consequences since it suggests thatthe small individual free energy components that comprise the overallfree energy of folding can be incrementally combined to optimize theyield of folded protein. TABLE 8 Factorial Experiment to Optimize theProtein Folding Yield for Immobilized His₆D(II)-FGFR1 at a final Gdn-HClconcentration of 0.38 M^(a) 7% Glycerol/ 500 mM NaCl 50 mM NaCl 50 mMNaCl pH 8.0 13.3%^(b) 9.3% 15.1% pH 8.0 13.6% 9.4% 15.8% pH 8.9 16.1%13.5% 17.8% pH 8.9 10.3% 17.8%

[0331] Results of a second round of refolding experiments at a finalGdn-HCl concentration of 0.09 M revealed that the Gdn-HCl is an evenmore important factor affecting the folding of His₆-D(II)FGFR1 (Table9). At pH 8.0 and 0.5 M NaCl, decreasing the Gdn-HCl concentration to0.09 M doubled the refolded protein yield, relative to the yieldobtained at pH 8.0, 0.5 M NaCl, and 0.38 M Gdn-HCl (Table 9). Inaccordance with the results obtained at a Gdn-HCl concentration of 0.38M, the yield of refolded His₆-D(II)-FGFR1 in 0.09 M Gdn-HCl was alsoincreased in the presence of glycerol. These results suggest that theimproved yield of refolded His₆D(II)-FGFR1 in glycerol (5 to 10%) andlower Gdn-HCl concentration are additive. Further, the results in Table9 reveal that the Hofmeister salt Na₂SO₄ increases the yield of refoldedprotein almost as well as 5 to 10% glycerol. TABLE 9 FactorialExperiment to Optimize the Protein Folding Yield for ImmobilizedHis₆-D(II)-FGFR1. Final Gdn-HCl of 0.09 M^(a) 5% 10% Glycerol Glycerol100 300 500 mM 50 mM 50 mM 50 mM mM mM NaCl NaCl NaCl NaCl Na₂SO₄ Na₂SO₄pH 8.0 25.6%^(b) 29.7% 36.5% 35.6% 32.2% 33.4%

[0332] Upon comparison of the biochemical conditions that increase theyield of refolded Ni²⁺NTA bound His₆-D(II)-FGFR1 (Tables 8 and 9) andthose conditions that increase the overall protein stability ofHis₆-D(II)-FGFR1 (FIGS. 19-24), it is clear that there is a strongcorrelation between the protein folding results and the proteinstability results. Glycerol, salts of the Hofmeister series, and pH 8.5to 8.9 improve protein folding yield and overall protein stability ofHis₆-D(II)-FGFR1.

[0333] These results are consistent with the model of protein folding inFIG. 26. If the aggregation of unfolded His₆-D(II)-FGFR1 is suppressedwhen immobilized to Ni²⁺NTA, and a simple two state equilibrium existsbetween U and N, then the factors that influence the relative positionof the equilibrium between U and N should be the same whether one startsfrom U (in the refolding experiment) or start from N (in the microplatethermal shift assay protein stability screen). Since thermodynamics arepath independent, only the initial and final states o f this reactionshould be important. Since similar biochemical conditions facilitateprotein stability and folded protein yield, the simple model for proteinfolding depicted in FIG. 26 is accurate for this protein. Thus, themicroplate thermal shift assay can serve as a rapid and general methodfor screening biochemical conditions that optimize protein folding.

EXAMPLE 20

[0334]FIG. 28 shows the results of microplate thermal shift assays ofusing each of four fluorescence probe molecules: bis-ANS, 2,6-TNS,1,8-TNS, and 2,6-ANS. Thrombin solution was diluted to 1 μM in 50 mMHepes, pH 7.5, and 0.1 M NaCl.

EXAMPLE 21 Comparison of Assay Results for a Fluorescence Scanner and aCharge Coupled Device Camera

[0335] A Gel Documentation and Analysis System (Alpha Innotech Corp.,San Leandro, Calif.) was used to perform a microplate thermal shiftassay. This system uses a CCD camera to detect fluorescence emissionfrom stained gels, dot blot assays, and 96 well plates. The excitatorylight source was a long wavelength UV trans-illumination box locateddirectly below the CCD camera. The 96 well plate to be assayed wasplaced on the trans-illumination box within the focal viewing area ofthe CCD camera (21×26 cm).

[0336] A 2 μM solution of human α-thrombin was prepared in 50 mM Hepes,pH 7.5, 0.1 M NaCl by diluting a 34 μM stock solution (1:17) of purifiedhuman α-thrombin (Enzyme Research Labs, Madison, Wis.). The humanα-thrombin solution also contained 100 μM 1,8-ANS. 100 μL of the humanα-thrombin-1,8-ANS solution was aliquoted into each of twelve wells of asingle row (row A) of a V-bottom polycarbonate microplate (Costar). Agradient block (RoboCycler™, Stratagene) was used to heat the twelvesamples, from 44 to 66° C., across the rows of the microplate. i.e. atemperature gradient of 2° C. per well was established. Thus, well A1was at 66° C. and well A12 was at 44° C. The control solution thatcontained 100 μM 1,8 ANS in the same buffer (no protein) was placed ineach of wells B1 to B 12. After adding a drop of mineral oil to eachwell to prevent evaporation, the plate was heated on the gradient blockfor 3 min. The contents of each well were then allowed to reach roomtemperature and transferred to a flat bottom microplate.

EXAMPLE 20

[0337]FIG. 28 shows the results of microplate thermal shift assays ofusing each of four fluorescence probe molecules: bis-ANS, 2,6-TNS,1,8-TNS, and 2,6-ANS. Thrombin solution was diluted to 1 μM in 50 mMHepes, pH 7.5, and 0.1 M NaCl.

EXAMPLE 21 Comparison of Assay Results for a Fluorescence Scanner and aCharge Coupled Device Camera

[0338] A Gel Documentation and Analysis System (Alpha Innotech Corp.,San Leandro, Calif.) was used to perform a microplate thermal shiftassay. This system uses a CCD camera to detect fluorescence emissionfrom stained gels, dot blot assays, and 96 well plates. The excitatorylight source was a long wavelength UV trans-illumination box locateddirectly below the CCD camera. The 96 well plate to be assayed wasplaced on the trans-illumination box within the focal viewing area ofthe CCD camera (21×26 cm).

[0339] A 2 μM solution of human α-thrombin was prepared in 50 mM Hepes,pH 7.5, 0.1 M NaCl by diluting a 34 μM stock solution (1:17) of purifiedhuman α-thrombin (Enzyme Research Labs, Madison, Wis.). The humanα-thrombin solution also contained 100 μM 1,8-ANS. 100 μL of the humanα-thrombin-1,8-ANS solution was aliquoted into each of twelve wells of asingle row (row A) of a V-bottom polycarbonate microplate (Costar). Agradient block (RoboCycler™, Stratagene) was used to heat the twelvesamples, from 44 to 66° C., across the rows of the microplate. i.e. atemperature gradient of 2° C. per well was established. Thus, well A1was at 66° C. and well A12 was at 44° C. The control solution thatcontained 100 μM 1,8 ANS in the same buffer (no protein) was placed ineach of wells B1 to B12. After adding a drop of mineral oil to each wellto prevent evaporation, the plate was heated on the gradient block for 3min. The contents of each well were then allowed to reach roomtemperature and transferred to a flat bottom microplate. In thisexperiment, no filters were employed to narrow the excitatory wavelengthto 7360 nm and the emission wavelength to ˜460 nm, which are optimalwavelengths for the 1,8 ANS fluorophore. The flat bottom plate was thenplaced on the near UV transillumination box and the CCD camera was usedto measure the amount of emitted light. The plate was also read using aconventional fluorescence plate reader (CytoFluor II), in order tocompare the results obtained by the two different detection methods. Theresults for the two detection methods are plotted in FIG. 40. Theresults in FIG. 40 show that the CCD camera is useful as a fluorescenceemission detector for monitoring the unfolding of a protein in themicroplate thermal shift assay.

EXAMPLE 22 Microplate Thermal Shift Assay Using a Charge Coupled DeviceCamera

[0340] An emission filter was used to block out all stray light outsidethe region of the emission region for 1,8-ANS (˜460 nm). In addition,the 5 μL miniaturized form of the microplate thermal shift assay wasemployed to test the CCD camera detection method in this configuration.Both the polycarbonate V-bottom and dimple plates were tested. Theexperiment was essentially the same as described in Example 16, exceptthat the volume of the assay was 5 μL in either the V-bottom or dimple96 well plates. The temperature range was 44 to 66° C. (right to left)for the V-bottom plate, and 46 to 70° C. (right to left) for the dimpleplate. Photographs of the CCD images are shown in FIG. 41. The V-bottomwell microplate image is shown in FIG. 41A. The dimple plate image isshown in FIG. 41B. The results obtained from the plate in FIG. 41A isshown in FIG. 42. The results in FIG. 42 show that data obtained using aCCD camera compare very well with data obtained using a fluorescenceplate reader that employs a photo-multiplier tube (PMT) for fluorescencedetection.

[0341] All publications and patents mentioned hereinabove are herebyincorporated in their entireties by reference.

[0342] While the foregoing invention has been described in some detailfor purposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention and appended claims.

1 3 1 4 PRT Artificial Sequence Description of Artificial Sequencearginine-p-nitroanilide 1 Ala Ala Pro Arg 1 2 6 PRT Artificial SequenceDescription of Artificial Sequence Six histidine epitope tag ofHis6D(II)-FGFR1 2 His His His His His His 1 5 3 6 PRT ArtificialSequence Description of Artificial Sequence C-terminal sequence ofHis6D(II)-FGFR1 3 Arg Arg Arg Arg Arg Arg 1 5

What is claimed is:
 1. An assay apparatus, comprising: a temperatureadjusting means for simultaneously adjusting a temperature of aplurality of samples in accordance with a predetermined temperatureprofile; and a receiving means for receiving spectral emission from thesamples while the temperature of the samples is adjusted in accordancewith the temperature profile.
 2. The apparatus of claim 1, wherein saidreceiving means receives fluorescent emission.
 3. The apparatus of claimor 1, wherein said receiving means receives ultraviolet light.
 4. Theapparatus of claim 1, wherein said receiving means receives visiblelight.
 5. The apparatus of claim 1, wherein said temperature adjustingmeans comprises: a temperature adjusting element for adjusting thetemperature of said heat conducting block.
 6. The apparatus of claim 1,wherein said temperature adjusting means comprises: a heat conductingblock; an adaptor disposed on said heat conducting block, wherein saidadaptor is configured to receive a container containing the plurality ofsamples; and a temperature adjusting element for adjusting thetemperature of said heat conducting block.
 7. The apparatus of claim 1,further comprising: a movable platform; wherein said temperatureadjusting means comprises a heat conducting block having a plurality ofwells formed therein, each of said plurality of wells configured toreceive a container containing one of the plurality of samples; whereinsaid movable platform is configured to receive a plurality of said heatconducting blocks; and a temperature adjusting element for adjusting thetemperature of said heat conducting block.
 8. The apparatus of claim 1,further comprising: a movable platform; wherein said temperatureadjusting means comprises a heat conducting block adapted to receive acontainer containing the plurality of samples, wherein said movableplatform is configured to receive a plurality of said heat conductingblocks; and a temperature adjusting element for adjusting thetemperature of said heat conducting block.
 9. The apparatus of claim 8,wherein said movable platform is a translatable platform.
 10. Theapparatus of claim 8, wherein said movable platform is a rotatableplatform.
 11. The apparatus of claim 1, wherein said receiving means isconfigured to receive spectral emission from the plurality of samplesone sample at a time.
 12. The apparatus of claim 1, wherein saidreceiving means is configured to simultaneously receive spectralemission from more than one sample of the plurality of samples.
 13. Theapparatus of claim 1, wherein said receiving means is configured tosimultaneously receive spectral emission from all of the plurality ofsamples.
 14. The apparatus of claim 6, wherein said temperatureadjusting means means further comprises: a temperature controller forchanging the temperature of said heat conducting block in accordancewith the pre-determined temperature profile.
 15. The apparatus of claim7, wherein said temperature adjusting means comprises: a temperaturecontroller for changing the temperature of said heat conducting block inaccordance with the pre-determined temperature profile.
 16. Theapparatus of claim 8, wherein said receiving means comprises: a lightsource for emitting an excitatory wavelength of light for the samples;and a sensor for detecting the spectral emission from the samples inresponse to the excitatory wavelength of light.
 17. The apparatus ofclaim 1, wherein said receiving means comprises a photomultiplier tube.18. The apparatus of claim 2, wherein said receiving means comprises afluorescence scanner.
 19. The apparatus of claim 2, wherein saidreceiving means comprises a fluorescence scanner.
 20. The apparatus ofclaim 11, wherein said receiving means comprises a fluorescence scanner.21. The apparatus of claim 12, wherein said receiving means comprises afluorescence scanner.
 22. The apparatus of claim 1, wherein saidreceiving means comprises a charge coupled device.
 23. The apparatus ofclaim 13, wherein said receiving means comprises a fluorescence imagingcamera.
 24. The apparatus of claim 22, wherein said receiving meanscomprises a CCD fluorescence imaging camera.
 25. The apparatus of claim1, wherein said receiving means comprises a diode array.
 26. An assayapparatus, comprising: a movable platform; a plurality of heatconducting blocks disposed on said platform, wherein each of saidplurality of heat conducting blocks is adapted to receive a plurality ofsamples; a light source for emitting an excitatory wavelength of lightfor the samples; a temperature adjusting means for adjusting thetemperature of said heat conducting blocks, thereby adjusting thetemperature of the samples; a sensor for detecting the spectral emissionfrom the samples in response to the excitatory wavelength of light; andwherein said movable platform is moved between heat conducting blocks tosequentially detect spectral emission from the samples in each of saidplurality of heat conducting blocks.
 27. The apparatus to claim 26,wherein said movable platform is a translatable platform.
 28. Theapparatus of claim 26, wherein said movable platform is a rotatableplatform.
 29. The apparatus of claim 26, wherein each of said pluralityof heat conducting blocks has a plurality of wells formed therein, eachof said plurality of wells configured to receive a container containingone of the plurality of samples.
 30. The apparatus of claim 26, whereineach of said plurality of heat conducting blocks is adapted to receive acontainer containing the plurality of samples.
 31. The apparatus ofclaim 26, wherein said temperature adjusting means comprises: atemperature controller for changing the temperature of said heatconducting blocks in accordance with a pre-determined temperatureprofile.
 32. The apparatus of claim 26, wherein said sensor comprises aphotomultiplier tube.
 33. The apparatus of claim 26, wherein said sensorcomprises a fluorescence scanner.
 34. The apparatus of claim 33, whereinsaid fluorescence scanner is configured to scan the plurality of samplesone sample at a time.
 35. The apparatus of claim 33, wherein saidfluorescence scanner is configured to simultaneously scan a subset of atleast two of the plurality of samples.
 36. The apparatus of claim 33,wherein said receiving means is configured to simultaneously receivespectral emission from all of the plurality of samples.
 37. Theapparatus of claim 36, wherein said receiving means comprises afluorescence imaging camera.
 38. The apparatus of claim 26, wherein saidsensor comprises a charge-coupled device.
 39. The apparatus of claim 38,wherein said sensor comprises a charge-coupled device camera.
 40. Theapparatus of claim 26, wherein said sensor comprises a diode array. 41.The apparatus of claim 37, wherein said fluorescence imaging camera isconfigured to simultaneously scan all of the plurality of samples in oneof said heat conducting blocks. 42 The apparatus of claim 37, whereinsaid fluorescence imaging camera is configured to simultaneously scanall of the plurality of samples in all of said plurality heat conductingblocks. 43 The apparatus of claim 39, wherein said charged coupleddevice camera is configured to simultaneously scan all of the pluralityof samples in one of said heat conducting blocks.
 44. The apparatus ofclaim 39, wherein said charge coupled device camera is configured tosimultaneously scan all of the plurality of samples in all of saidplurality heat conducting blocks.
 45. The apparatus of claim 26, whereinat least one sample of the plurality of samples comprises a biologicalpolymer.
 46. The apparatus of claim 26, wherein at least one sample ofthe plurality of samples comprises a protein.
 47. The apparatus of claim26, wherein at least one sample of the plurality of samples comprises anucleic acid.
 48. The apparatus of claim 1, further comprising: acomputer controller for controlling the operation of said temperatureadjusting means.
 49. The apparatus of claim 14, wherein said temperaturecontroller comprises a processor.
 50. The apparatus of claim 15, whereinsaid temperature controller comprises a processor.
 51. The apparatus ofclaim 31, wherein said temperature controller comprises a processor. 52.The apparatus of claim 26, wherein said temperature adjusting meansindependently adjusts the temperature of each of said heat conductingblocks.
 53. An assay apparatus, comprising: a heating means forsimultaneously heating a plurality of samples; and a receiving means forreceiving spectral emission from the samples while the samples are beingheated.